In situ heat induced antigen recovery and staining apparatus and method

ABSTRACT

Contemplated herein is an automated microscope slide antigen recovery and staining apparatus and method that features a plurality of individually operable miniaturized pressurizable reaction compartments for individually and independently processing a plurality of individual microscope slides. The apparatus preferably features independently movable slide support elements each having an individually heatable heating plate. Each slide support element preferably supports a single microscope slide. Each microscope slide can be enclosed within an individual pressurizable reaction compartment. Pressures exceeding 1 atm or below 1 atm can be created and maintained in the reaction compartment prior to, during or after heating of the slide begins. Because of the ability to pressurize and regulate pressure within the reaction compartment, and to individually heat each slide, each slide and a liquid solution or reagent thereon can be heated to temperatures that could not be obtained without the enclosed pressurized environment of the reaction compartment. A reagent dispensing strip having a plurality of reconfigurable reagent modules may also be used.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/439,722, filed May 24, 2006, now U.S. Pat. No. 7,897,106, whichclaims benefit under 35 U.S.C. 119(e) of each of U.S. ProvisionalApplication Ser. No. 60/684,047 filed May 24, 2005, U.S. ProvisionalApplication Ser. No. 60/689,386 filed Jun. 10, 2005, and U.S.Provisional Application Ser. No. 60/730,744, filed Oct. 27, 2005. Eachof the applications listed above is hereby expressly incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

BACKGROUND

The present invention is related to the field of treating samples onmicroscope slides including analytical substrates, and more specificallyto the field of heat induced antigen recovery and staining.

In anatomical pathology labs (e.g., histology, cytology) it is knownthat certain immunohistochemical procedures, herein known as IHC assays,are performed on biological specimens including, for example,formalin-fixed paraffin-embedded tissues and cell preps. Also used inthe art are several IHC antibodies (abs) like Estrogen receptor abs,Progesterone receptor abs, Proliferation abs like Ki-67, which requirethe use of high temperature unmasking techniques, (i.e., antigenretrieval, high temperature epitope recovery, and antigen unmasking),prior to application of the antibody for labeling cell structures(antigens).

There are several procedures known in the art for the “unmasking” ofantigens that have been rendered “hidden” by formalin fixation.Procedures known in the art include treating the biological specimen inaqueous solutions (e.g., water) that may include buffers (e.g., citrate,EDTA, urea, etc.), along with detergents or surfactants (e.g., Brij 35,Tween, SDS, NP-40 and Igepal). These known formulations are heated totemperatures from around 60° C. to about 120° C. These heatedformulations are in contact with the biological specimen for variousamounts of time (e.g., about 10 minutes to about 90 minutes) therebycausing the “masked” antigen to become “unmasked” so the antibodies usedin the IHC assays can attach to their corresponding antigens which areassociated with the biological specimen.

Types of apparatuses that are known and used to perform the heating ofthe antigen retrieval solutions and the biological specimen includewaterbaths, steamers, pressure cookers, autoclaves, microwave ovens andconvection ovens. Since water boils at 100° C. at normal atmosphericpressure, antigen retrieval solutions even with other chemicals presenthave only been able to reach temperature from about 98° C. to 100° C.before evaporative heat loss inhibits the solution from reaching highertemperatures. Pressure cookers and autoclaves overcome this by allowingfor pressurization of the solutions so higher temperatures can beachieved without evaporation of the heated fluid. Since there areantibodies that require the antigen retrieval solution be attemperatures exceeding 100° C., many laboratories must use pressurecookers to heat the biological specimen with its antigen retrievalsolution to attain temperatures up to 120° C., without which the antigenwould not be “unmasked” preventing the antibody from binding to theantigen.

There remains a need for an apparatus able to produce high temperaturepressure conditions for single slides being subjected to individualizedantigen retrieval conditions without relying on clumsy and unwieldydevices such as pressure cookers and autoclaves.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top plan view of a reagent strip (reagent dispensing strip)of the present invention.

FIG. 2 is a cross-sectional side view taken through line 1-1 of thereagent dispensing strip of FIG. 1.

FIG. 3 is a cross-sectional view along line 3-3 of FIG. 2.

FIG. 4 is a side view of the reagent strip of FIG. 1 having two reagentmodules detached therefrom.

FIG. 5 is a bottom plan view of the reagent strip of FIG. 1 with areagent module reattached via a tile connector.

FIG. 6 is a top plan view (A), side view (B), and bottom plan view (C)of the tile connector used in FIG. 5.

FIG. 7 is a bottom plan view of a reagent strip with two reagent modulesreconnected via a pair of tile connectors.

FIG. 8 is a cross-sectional view taken through line 8-8 of a reagentmodule and tile connector.

FIG. 9 is a top plan view of a base of another reagent strip of thepresent invention.

FIG. 10 is a top plan view of the reagent strip of FIG. 9 with reagentcontainers disposed upon the base.

FIG. 11 is a cross-sectional side view taken through line 11-11 of FIG.10.

FIG. 12 is a cross-sectional view taken through line 12-12 which showshow a reagent container fits onto or is removed from the base of thereagent strip of FIG. 11.

FIG. 13 is a side view of the reagent strip of FIG. 11.

FIG. 14 is a front end view of the reagent strip of FIG. 13.

FIG. 15 is a bottom plan view of the reagent strip of FIG. 10 after the“G” reagent container has been removed and the “H” reagent container hasbeen moved into the former “G” position.

FIG. 16 is a bottom plan view of the reagent strip of FIG. 10 after the“G” reagent container has been replaced with a “G-2” reagent container.

FIG. 17 is a top plan view of another reagent strip of the presentinvention which has exchangeable reagent modules.

FIG. 18 is a cross-sectional view taken through line 18-18 of FIG. 17.

FIG. 19 is a side view of the reagent strip of FIG. 17.

FIG. 20 is a front end view of the reagent strip of FIG. 19.

FIG. 21 is a bottom plan view of the reagent strip of FIG. 17 afterreagent module “G” has been removed.

FIG. 22 is a bottom plan view of the reagent strip of FIG. 17 after thereagent module “G” has been exchanged with reagent module “G-2”.

FIG. 23 is a cross-sectional side view of a reaction module (reactioncompartment, slide, support element, and reagent strip support device)of the present invention before the reagent strip has been inserted intothe reagent strip support device, and before a microscope slide has beendisposed on the slide support element.

FIG. 24A is a cross-sectional side view of the reaction module of FIG.23 in operation in a reagent dispensing phase.

FIG. 24B is a transverse cross-sectional view of the reaction module ofFIG. 24A.

FIG. 25A is a cross-sectional side view of the reaction module of FIG.23 and FIG. 24A in a reagent drainage phase.

FIG. 25B is a transverse cross-sectional view of the reaction module ofFIG. 25A.

FIG. 26A is a cross-sectional side view of the reaction module of FIG.25A in a rinse buffer dispensing phase.

FIG. 26B is a transverse cross-sectional view of the reaction module ofFIG. 26A.

FIG. 27A is a cross-sectional side view of the reaction module of FIG.26A in a rinse buffer drainage phase.

FIG. 27B is a transverse cross-sectional side view of the reactionmodule of FIG. 27A.

FIG. 28 is a cross-sectional view of the reaction module of FIGS. 23-27Bafter the reagent strip is completely used and the microscope slide isremoved from the slide support element.

FIG. 29 is an enlarged version of FIG. 24A.

FIG. 30 is an enlarged version of FIG. 26A.

FIG. 31A is a cross-sectional top view of the reaction compartment andslide support element of the reaction module of FIG. 29 which shows aclockwise air mixing step.

FIG. 31B is a transverse cross-sectional view of the air ports of theslide support element of FIG. 31A.

FIG. 32A is a cross-sectional top view of the reaction compartment andslide support element of the reaction module of FIG. 29 which shows acounterclockwise air mixing step.

FIG. 32B is a transverse cross-sectional view of the air ports of theslide support element of FIG. 32A.

FIG. 33 is a view of the slide and detached components of the heatingelement of the slide support element of FIG. 28.

FIG. 34A is a cross-sectional top view of a slide support of a reactionmodule element with the slide and heating element detached to show airflow through the air cooling ducts which are used to enhance a rapidcooling of the heating element.

FIG. 34B is a transverse cross-sectional view through the air coolingducts of the slide support element of FIG. 34A.

FIG. 35A is a cross-sectional side view of the reaction module of FIG.34A.

FIG. 35B is a transverse cross-sectional view through the air coolingducts of the slide support element of FIG. 35A.

FIG. 36 is a view of the slide and detached components of the heatingelement of the slide support element of FIG. 28.

FIG. 37A is a cross-sectional top view of a slide support of a reactionmodule element with the slide and heating element detached to show airflow through the air cooling ducts which are used to rapidly cool theheating element.

FIG. 37B is a transverse cross-sectional view through the air coolingducts of the slide support element of 37A.

FIG. 38A is a cross-sectional side view of the reaction module of FIG.34A.

FIG. 38B is a transverse cross-sectional view through the air coolingducts of the slide support element of FIG. 38B.

FIG. 39 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having ventilation holes.

FIG. 40 is a cross-sectional view of the reagent strip of FIG. 39 takenthrough line 40.

FIG. 41 is a side view of the reagent strip of FIG. 39.

FIG. 42 is a bottom plan view of the reagent strip of FIG. 39.

FIG. 43 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having ventilation holes.

FIG. 44 is a cross-sectional view of the reagent strip of FIG. 43 takenthrough line 44.

FIG. 45 is a side view of the reagent strip of FIG. 43.

FIG. 46 is a bottom plan view of the reagent strip of FIG. 43.

FIG. 47 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having ventilation holes.

FIG. 48 is a cross-sectional view of the reagent strip of FIG. 47 takenthrough line 48.

FIG. 49 is a side view of the reagent strip of FIG. 47.

FIG. 50 is a bottom plan view of the reagent strip of FIG. 47.

FIG. 51 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having a ventilation slot.

FIG. 52 is a cross-sectional view of the reagent strip of FIG. 51 takenthrough line 52.

FIG. 53 is a side view of the reagent strip of FIG. 51.

FIG. 54 is a bottom plan view of the reagent strip of FIG. 51.

FIG. 55 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having a ventilation slotand a rapid cooling window.

FIG. 56 is a cross-sectional view of the reagent strip of FIG. 55 takenthrough line 56.

FIG. 57 is a side view of the reagent strip of FIG. 55.

FIG. 58 is a bottom plan view of the reagent strip of FIG. 55.

FIG. 59 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having a ventilation slotand a rapid cooling window.

FIG. 60 is a cross-sectional view of the reagent strip of FIG. 59 takenthrough line 60.

FIG. 61 is a side view of the reagent strip of FIG. 59.

FIG. 62 is a bottom plan view of the reagent strip of FIG. 59.

FIG. 63 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having a ventilation slot.

FIG. 64 is a cross-sectional view of the reagent strip of FIG. 63 takenthrough line 64.

FIG. 65 is a side view of the reagent strip of FIG. 63.

FIG. 66 is a bottom plan view of the reagent strip of FIG. 63.

FIG. 67 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having a rapid coolingwindow.

FIG. 68 is a cross-sectional view of the reagent strip of FIG. 67 takenthrough line 68.

FIG. 69 is a side view of the reagent strip of FIG. 67.

FIG. 70 is a bottom plan view of the reagent strip of FIG. 67.

FIG. 71 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having a ventilation slotand a rapid cooling window.

FIG. 72 is a cross-sectional view of the reagent strip of FIG. 71 takenthrough line 72.

FIG. 73 is a side view of the reagent strip of FIG. 71.

FIG. 74 is a bottom plan view of the reagent strip of FIG. 71.

FIG. 75 is a top plan view of an alternate version of the reagent stripof the present invention, the reagent strip having ventilation holes anda rapid cooling window.

FIG. 76 is a cross-sectional view of the reagent strip of FIG. 75 takenthrough line 76.

FIG. 77 is a side view of the reagent strip of FIG. 75.

FIG. 78 is a bottom plan view of the reagent strip of FIG. 75.

FIG. 79 is a cross-sectional side view of an alternate embodiment of areaction module of the present invention.

FIG. 80 is a cross-sectional side view of the reaction module of FIG. 79in an alternate processing configuration.

FIG. 81 is a cross-sectional side view of the reaction module of FIG. 79in an alternate processing configuration.

FIG. 82 is a cross-sectional side view of the reaction module of FIG. 79in an alternate processing configuration.

FIG. 83 is a cross-sectional side view of the reaction module of FIG. 79in an alternate processing configuration.

FIG. 84 is an enlarged fragmented cross-sectional side view of thereaction module of FIG. 79 in an alternate processing configuration.

FIG. 85 is a front cross-sectional view of a plurality of reactionmodules of the present invention combined in a chamber to form an insitu antigen recovery and staining apparatus of the present inventionwherein the reaction modules are in various phases of operation.

DETAILED DESCRIPTION OF THE INVENTION

Contemplated herein is an automated microscope slide staining apparatusthat features a plurality of individually operable miniaturizedpressurizable reaction compartments for individually and independentlyprocessing a plurality of individual microscope slides (where usedherein the term “microscope slide” is also intended to refer to othermicroscopy analytical devices which are used as vessels or supportstructures for supporting biological and biochemical specimens fortesting and analysis, and which are sized and shaped to fit within areaction compartment as described and contemplated herein and whichinclude, but are not limited to, test tubes, petri dishes, andmicroarray plates, as well as standard microscope slides). The automatedin-situ antigen recovery and staining apparatus of the present inventionpreferably features independently movable slide support elements eachhaving an individually heatable heating plate. Each slide supportelement preferably supports a single microscope slide. Each slidesupport element with the microscope slide thereon is enclosed within itsown individual pressurizable reaction compartment and/or comprises aportion thereof. In one treatment step, antigen retrieval solution isdisposed on the slide and the heating plate heats the slide and theantigen retrieval solution thereon to temperatures of up to and in somecases exceeding 150° C. by regulating the pressure within the reactioncompartment thereby increasing the temperature that the solution canattain. In one embodiment each reaction compartment has its ownindividual pressure regulator, device, or switch to regulate pressurewithin the reaction compartment. Pressures exceeding 1 atm (i.e.,exceeding 14.7 psi, 0 psig or 101.325 kPa) or below 1 atm can be createdand maintained in the reaction compartment. The reaction compartment canhold, for example, 0.1 ml to 100 ml of antigen retrieval solution.

Where used herein the term “biological specimen” includes, but is notlimited to, unprocessed specimens, processed specimens, paraffinembedded tissue, whole mounts, frozen sections, cell preps, cellsuspensions, touch preps, thin preps, cytospins, and other biologicalmaterials or molecules including blood, urine, cerebrospinal fluids,pleural fluids, ascites fluids, biopsy materials, fine needle aspirates,pap smears, swabbed cells or tissues, microbiological preps includingbacteria, viruses, parasites, protozoans, biochemicals including, butnot limited to proteins, DNA, RNA, carbohydrates, lipids, microarrays,ELISA reagents and analytes, synthetic macromolecules, phospholipids,support structures of biological molecules (e.g., metals, beads,plastics, polymers, glass), or any other materials attached to abiological testing substrate for processing, examination, orobservation.

Because of the ability to pressurize and regulate pressure within thereaction compartment, and to individually heat each slide, each slideand a liquid solution or reagent thereon (e.g., including, but notlimited to, antigen retrieval reagents, RNA and DNA probes, citratebuffer, EDTA, TRIS, PBS, with or without surfactants or detergents likeSDS, Tween, Brij, ionic and non ionic detergents, and siliconeadditives, rinse buffers, immunohistochemical reagents, histochemicalreagents, in-situ hybridization reagents, PCR reagents, coverslippingreagents, silicone oils, mineral oils, detection reagents and processingreagents, liquid reagents, reconstituted dry reagents, biologicalreagents and aqueous and non-aqueous reagents, and deparaffinizingcompositions of water with one or more silicone surfactants or siliconeadditives) can be heated to temperatures that could not be obtainedwithout the enclosed pressurized environment of the reactioncompartment. For example, since the vapor produced by the solution iscontained in the reaction compartment (or is released in a regulatedmanner), the pressure in the reaction compartment can be regulated toproduce a temperature required by the user. Pressures (“negativepressure”, i.e., vacuums) below 1 atm (i.e., below 14.7 psi, 0 psig or101.325 kPa) may also be created and maintained within the reactioncompartment. For example, vacuum pressures of from 100 kPa to 10 kPa to1 kPa to 100 Pa to 10 Pa to 1 Pa to 0.1 Pa may be formed and held in thereaction compartment.

Each reaction compartment of each reaction module can be heatedseparately and independently from the other reaction compartments by aconductive heating element (or heating plate) underneath or adjacent tothe slide (e.g., wherein the heating element is in a sidewall of thereaction compartment or in a cavity). In a preferred embodiment, asnoted above, the enclosed reaction compartment containing at least aportion of the microscope slide has an antigen retrieval solutiondeposited onto the slide in the reaction compartment. The slide is thenheated, in a preferred embodiment, to a temperature of about 100°C.-300° C. and under a pressure from 0.1 psig (102.016 kPa) to 350 psig(2514 kPa). In one embodiment the containment of the pressure isproportional to the temperature of the antigen retrieval solution, suchthat the regulation of both the temperature of the heating element ofthe reaction compartment and the regulation of the pressure generated bythe solution on the slide can be adjusted during the automated stainingprocedure.

In one example, the heating element could heat the slide to at least120° C. and the pressure in the reaction compartment would be 16 psigwherein, the solution on the slide in contact with the biologicalspecimen would be about 130° C. It would be apparent to one of ordinaryskill in the art of pressure regulated vessels that the temperatureattained by the antigen retrieval solution would be dependent on theregulation of either the pressure generated or the temperature of theheating element or both. If regulation of the temperature of thesolution is to be determined by the pressure, the heating plate can beset at 130° C. (for example) and the pressure relief valve could be setto a level to maintain a pressure of 19 psig (232.3 kPa) within thereaction compartment, for example. Thus, the temperature of the antigenretrieval solution would not exceed 130° C. and would remain in therange of 120° C.-130° C.

If regulation of the temperature of the solution is determined bytemperature of the heating element, then the heating plate can beregulated to heat up to a desired temperature. Once the desired pressurewithin the reaction compartment has been reached, the heating elementpressurizing means are adjusted to keep the desired pressure within thepreferred limits. The reaction compartment under some conditions doesnot necessarily require a pressure regulator since the pressure in thereaction compartment was regulated solely by the temperature of theheating element. In some embodiments it would be advantageous to have aregulator to relieve pressure if the pressure exceeds desired levels orto have a pressure regulator which would cause the heating element to beturned on and off depending on the desired pressure.

Since boiling of the solution on the slide is suppressed by thecontainment of the pressure, the antigen recovery buffer in the reactioncompartment may appear not to be boiling even though it has actuallyreached a temperature above 100° C. Elimination or reduction of vaporloss due to boiling is advantageous because it removes the necessity ofadding additional buffer during processing (such as is necessary whenusing certain other apparatuses known in the art, e.g., as shown in U.S.Pat. No. 5,654,200; U.S. Pat. No. 5,595,707; U.S. Pat. No. 6,296,809;U.S. Pat. No. 6,352,861; U.S. Pat. No. 6,582,962; U.S. Pat. No.6,096,271; U.S. Pat. No. 6,180,061; U.S. Pat. No. 6,183,693; U.S. Pat.No. 6,541,261; or U.S. Pat. No. 6,783,733) even when only small amountsof buffers are initially used (e.g., 500 μl-4 ml) and treatment timesare extended up to 60 minutes at high temperatures (e.g., 130° C.). Lossof antigen retrieval volume in the present invention is minimal due tocontainment of vapors generated. Another important advantage tominimization of boiling at high temperatures is that the biologicalspecimen on the slide is not subjected to extreme agitation from bubblesbeing formed which could cause the biological specimen to detach fromthe glass slide or be otherwise damaged. Moreover, the controlledpressurized micro-environment in the reaction compartment of the presentinvention is very efficient because the amount of buffer that is used isminimal and the amount of time needed to heat to high heat conditions(e.g., 120° C.-140° C.) is also minimal (e.g., 5 minutes).

Commercial pressure cookers which are currently available for use inantigen retrieval procedures are bulky and require a greater amount ofbuffer and time to complete the high temperature antigen retrievalprocess and furthermore must be used to treat many slides in the samecontainer. The typical pressure cooker procedure from start time to thelast step (rinse) typically lasts 45-60 minutes. Only a few differentbuffers can be heated at the same time, (on the order of 5-6 separateslide treatment containers) within a pressure cooker's main reactioncompartment. Each separate slide container in a commercial pressurecooker requires significant volumes of antigen retrieval solution (e.g.,45-50 mls per container). As opposed to the pressure cookers which areused in the field of antigen retrieval, the apparatus and method of thepresent invention preferably uses vapor from the reagent on the slideitself to produce an elevated pressure in the individual reactioncompartment. Pressure cookers, to the contrary, rely on a separateliquid present within the bottom of the vessel to produce the vapornecessary to cause increased pressure within the vessel for inducingantigen retrieval on the slides therein. This method requires theadditional step of heating the separate liquid to an elevatedtemperature before the process of heating the slide and the reagentthereon can begin.

Each of the individual reaction compartments of the apparatus of thepresent invention, to the contrary, utilize relatively small quantitiesof antigen retrieval buffer (e.g., 0.5-5 ml per slide) and heat upquickly and cool quickly due to the small amounts of liquid and area tobe heated and cooled. Even a volume of 0.1-1 ml per slide can be usedwith the present invention and the typical time from start to finishusing the present invention can be as low as 20 minutes, for example.

In a preferred embodiment of the invention, to maintain small amounts ofliquid reagents (e.g., including, but not limited to antigen retrievalreagents, RNA and DNA probes, citrate buffer, EDTA, TRIS, PBS, with orwithout surfactants or detergents like SDS, Tween, Brij, ionic and nonionic detergents, and silicone additives, rinse buffers,immunohistochemical reagents, histochemical reagents, in-situhybridization reagents, PCR reagents, coverslipping reagents, siliconeoils, mineral oils, detection reagents and processing reagents, liquidreagents, reconstituted dry reagents, biological reagents and aqueousand non-aqueous reagents, and deparaffinizing compositions of water withone or more silicone surfactants or silicone additives) from beingreduced in volume by the conversion from a liquid phase to a gaseousphase, and loss thereof, during heating (as occurs in other commerciallyavailable systems), the reaction compartment, when closed, can bepre-pressurized, individually, prior to the heating of the slide andreagent. This pre-pressurization from a separate pressurization source,(i.e., rather than solely from the vapor pressure produced by the heatedliquid), can significantly reduce the amount of loss of the gaseousphase (evaporation) of small amounts of liquid reagents (e.g., 100 μl-4ml) under high temperature conditions (e.g., 100° C.-140° C.) forextended heating times (e.g., 10-60 minutes), thereby eliminating therequirement of adding additional reagent after the treatment process hasbegun. For example, preferably, 0.1-4 milliliters of reagent (e.g.,antigen retrieval reagent) is placed on the slide, the reactioncompartment is then pre-pressurized and then the heating element beginsto heat the reagent. The pre-pressurization of the reaction compartment,followed by heating of the reagent, produces an environment for thereagent to reach temperatures exceeding 100° C., for example up to 140°C., with minimal reagent loss due to gas phase formation (evaporation).

As is apparent from the above example, the temperatures and pressurescould alternatively be set for any desired level for any protocol knownin the art of staining biological specimens. Super high temperatureconditions can also be achieved using the present invention. These superhigh heating conditions can reach and exceed 350° C. and 300 psig (2170kPa) due to pressurization, pre-pressurization, and the particularconstruction of the reaction compartment (described below). Theindividual pre-pressurizable reaction compartments of the presentinvention can hold any type of vessel or substrate known in the art forcontaining a biological specimen for testing. Vessels or substratesinclude but are not limited to glass and plastic microscope slides,micro titer plates, tubes, flasks, micro arrays, vials, plates, andother vessels for containing biological materials.

In a preferred embodiment, the reaction compartment can bepre-pressurized and remain pressurized even under very high pressures ofover 300 psig (2170 kPa) to produce very high temperatures exceeding300° C. for use in special procedures that require such very hightemperature conditions. In alternate embodiments, the reactioncompartment can generate and sustain temperatures and pressures, forhigh heat conditions, in the range of 100 to 150° C. to 200° C.-250° C.to 300° C. Preferably, a pressure of at least 15 psig (204.7 kPa) ismaintained within the reaction compartment during heating.

As described elsewhere herein, this heat can be generated by aconductive heating element positioned beneath the microscope slide, aconductive heating element in the reaction compartment wall, other typesof heating devices in locations adjacent to the reagents being heated,and microwaves passing through the walls of the reaction compartment toheat the regents, for example. These types of heating devices can all beincorporated separately or together with the systems described hereinfor the regulation of pressure.

In a preferred embodiment, the regulation of pressure within thereaction compartment, either by pre-pressurization or by pressureproduced by evaporation of the heated reagent, is a critical componentof the invention.

In a preferred embodiment the present invention eliminates the use of asingle large container (e.g., a pressure cooker) to treat one or aplurality of slides under pressure. Each individually operable reactioncompartment of the apparatus of the present invention can treat anindividual slide disposed therein with an individualized reagent at anindividualized temperature and pressure without relying on or affectingany of the other plurality of slides in their respective reactioncompartments in the same apparatus, i.e., each reaction compartment canoperate independently of each other reaction compartment. The advantageof the present invention is in its ability to treat every slide in theinstrument separately and independently at an individualized temperatureand pressure without reliance on any other processing devices of theother reaction compartments thereby increasing efficiency in theproduction and processing of specimens and providing a constant workflowadvantage. Using the present invention, a technician can separatelybegin a test of a slide utilizing any protocol at any temperature orpressure without affecting or stopping the other reaction compartmenteven when those other reaction compartments are already in use.

As described above, the temperature of the reagent on or in the slide(or vessel) in the reaction compartment can be maintained by regulatingthe temperature of the heating element or by regulating the pressure bya pressure regulator or by both in combination. In one embodiment, forexample, the reaction compartment can be pre-pressurized to 23 psig(259.9 kPa), the heating element can be set to reach 125° C., and themaintenance pressure can be set to 23 psig (259.9 kPa), wherein thereagent on the slide reaches a temperature of 125° C. for 10 minutes,and is then cooled for further processing. In a preferred embodiment,the pre-pressurized conditions are defined as “separately pressurizing areaction compartment that has a biological specimen contained therein.”In this embodiment, as noted above, the pressure is not produced by thevaporization of the liquid contained in the reaction compartment, butrather by a separate pressurization system or device. The reactioncompartment can hold one individual biological specimen or can also holdseveral biological specimens. In the preferred embodiment, an individualreaction compartment is pre-pressurizable and is constructed to containonly one slide having a biological specimen thereon.

Without wishing to be held to theory, the pre-pressurization process,when incubating reagents (including any reagents described elsewhereherein) features conditions to minimize evaporative loss of reagents andor aqueous phase (water) or oil phase (oil) of reagents during heatingand/or ambient temperature staining conditions. A further aspect of theembodiment featuring the individual pre-pressurized reaction compartmentis that during the reaction process, pressure causes the reagents tocome in close, intimate contact with the biological specimen by being“pressed” against the biological specimen wherein the physical contactbetween them is increased due to the pressure exerted on the reagent andthereby upon the biological specimen.

This pressurized force of the reagent toward the biological specimenpreferably decreases the time of treatment by the reagents due to veryefficient contact of the reagents with the biological specimen.Specimens may have their processing times significantly reduced due tosuperior staining caused by the reagents being “pressed” against thebiological specimen, thus enhancing intimate contact with the biologicalspecimen.

Polymerase Chain Reaction (PCR), including tissue PCR is dependent onthe retention of the water levels in the reagents during processing.Specific water concentrations, pH conditions, and temperatures have tobe strictly met in order for the PCR reaction to be successful. Thepressurized conditions of the reaction compartment of the presentinvention are ideal for these conditions (low evaporation) to be metduring staining. This low evaporation, due to an individual pressurizedmicro-environment (the individual reaction compartment) is ideal for PCRreactions on glass microscope slides, plastic microscope slides,vessels, tubes, micro arrays, micro titer plates, plates, or any othervessel used for the containment of biological specimens. Thispressurization can also be used at ambient temperature as well (e.g.,25° C.).

The preferred embodiment of the pre-pressurized reaction compartmentincludes not only individual reaction compartments that hold only onebiological containing vessel or slide, but also a pre-pressurizedchamber that can hold several vessels or slides that can bepre-pressurized to decrease processing time and reduce evaporation orreagent loss.

In a further embodiment, the heating of the reagent on the slide can bedone by pre-pressurizing the reaction compartment with heated (below100° C.) or super heated (above 100° C.) air that would maintain therequired temperature for the protocol or would at least pre-heat thereaction compartment prior to the heating element reaching heatingtemperature or being turned on to heat, and maintain the heating of thereagent on the microscope slide.

In a particularly preferred embodiment of the invention, one or more ofthe reaction compartments of the apparatus are pre-pressurized aftermicroscope slides are enclosed therein. The pre-pressurization of thereaction compartment may occur before, during, or after the heatingelement is actuated to heat the microscope slide and reagent thereon.

In another embodiment of the invention, a plurality of slides togetherin a common chamber may be pre-pressurized and heated therebyeliminating the need to add additional reagent to each slide during theantigen retrieval process. For example, the plurality of slides in theapparatuses shown in U.S. Pat. Nos. 5,654,200; 5,595,707; 6,296,809;6,352,861; 6,582,962; 6,096,271; 6,180,061; 6,183,693; 6,541,261; or6,783,733 may be enclosed within a pressurizable chamber andpre-pressurized before, during, or after the heating step begins.

In a preferred embodiment of the invention a plurality of slides areenclosed within a common chamber, reagent is applied to the slides(before or after enclosure within the chamber), the chamber ispressurized to a level above atmospheric pressure, and the slides areheated so the temperature of the reagent on the slide exceeds 85° C. andmore preferably exceeds 100° C. Further, the reagent could be applied tothe slides after the chamber is pressurized.

The same steps as above could be followed in an alternate embodimentabsent inclusion of a heating process. The result of the process withoutheating is reduced evaporation or vaporization of the reagent from theslide while reagent is reacting with the specimen or sample on the slideand an increase in the physical interaction thereof, due to increasedpressure of the reagent with the specimen or sample on the slide.

In the preferred embodiment, each microscope slide is processed withinits own individual reaction compartment that can be individuallypressurized. Each reaction compartment is separate from every otherreaction compartment which together comprise an automated slide stainingapparatus to process a plurality of slides simultaneously, if desired,yet individually. Each reaction compartment is functionally independent(i.e., non-interdependent) from each other reaction compartment. Theindependent operability of each reaction compartment is due to eachreaction compartment having separate operational mechanisms, includingbut not limited to, individually moving slide support elements,individually moving reagent dispensing strips, and individually movableor stationary ports and dispensers for rinses, pressure, vacuum andwaste disposal. No single individual processing device in any of thereaction compartments is dependant at anytime on the operation of theprocessing components of another reaction compartment whether it is inoperation or not, including, preferably, microprocessing programs uniqueto each reaction compartment. All processing components (e.g.,including, but not limited to, reagent dispensers, rinses ports, vacuumports, pressure ports, waste ports, mixing ports, slide supports,reaction compartments, air cooling ducts, and liquid cooling ducts) canbe individually and independently moveable and/or usable.

In a further embodiment of the present invention, the microprocessor ofthe invention utilizes an operating system that can have multiple,individually, and/or simultaneously running processing programs,partially or completely specific to each individual reactioncompartment. This would enable a simple approach to programming byeliminating the need for the microprocessor to have one operatingprogram to determine the status of all processing steps as on currentslide staining instruments (e.g., as shown in U.S. Pat. Nos. 5,439,649,5,595,707, 5,758,033, 5,839,091, 6,296,809, 6,352,861 and 6,783,733). Instaining instruments known in the prior art, microprocessors have aprocessing program which is aware of all the steps for each slide in thestaining process and which determines the correct time to activate acommon processing device for a particular slide's use (i.e.—reagentdispenser, rinses, air applications, etc.) This “thinking and reacting”approach to the computer's involvement in processing a plurality ofslides is inefficient. A lagtime is produced when all the slides areunder the control of one program. This inefficient use of time causesincreased time for processing just because of the requirement of themicroprocessor to determine the next step for each slide and determineany conflicts with two or more slides needing to be processed by acommon device at the same time. This type of microprocessing delays thecompletion of the processing of a slide that would need a processingdevice at the same time as another slide or multiple slides.

Some staining instruments known in the art feature a “STAT RUN” option.With this type of processing, the user has already started a stainingrun and has decided that one or more additional slides need to be placedon the instrument and processed because the processing of the“additional slides” is more urgent. The user can put the “original”slides on a lesser priority setting. The “new slides” can then be placedon the instrument and would receive the priority use of the “new slides”of all the processing devices. In between the priority stainingprotocol, the processing devices can then be used to treat the“original” or “non stat” slides that were on the instrument initially.The requirement for this type of interrupted processing is eliminateddue to the features of the present invention.

The advantages of the present invention microprocessor having a singleor unique program for each reaction compartment program eliminates theneed for a microprocessor which is able to plan the interdependent stepsfor a plurality of slides being processed, as required by prior artsystems. A further advantage of having a separate microprocessingprogram unique to each reaction compartment, is that if themicroprocessors of one or several reaction compartments failed, therewould be no effect on the operation of the other reaction compartments.One advantage to this system of microprocessing is that there is noappreciable downtime in the event of a microprocessor failure in one ora few reaction compartments. To the contrary, in the instruments of theprior art, if the microprocessor or operating system fails, then theinstrument is completely inoperable and must be repaired.

In the present invention, in a preferred embodiment, there can be acommon “master” operating system that could be in communication witheach individually unique program so that the user can open a separateprogram specific to any or all of the reaction compartments at anytime.The separate individual program running a specific reaction compartmentwould have all the protocols loaded therein for completely processing aslide. The separate program could be updated and edited by the user andwith the help of the master program could update all the other separateprograms so that each reaction compartment could have the same protocolsupdates or edits. In the event of a master program failure, the separateunique programs to each reaction compartment would still be operationalto process slides; it just would lose the ability of communicate withthe separate programs of the other reaction compartments for updating,downloading, or uploading information. In a variation, each reactioncompartment may be individually separated and unique to itself inregards to its operating program with no link to the other reactioncompartments. A further advantage to having a master operating system isthe ability to communicate with the other separate reaction compartmentprograms for diagnostic purposes, uploading, downloading, and generaland specific communications between reaction compartments.

In one embodiment of the present invention, all the motion controlrequirement necessary for operation of the system can be in the form ofAC, DC, solar, and optionally other power sources like pneumatic andsteam. The microprocessor can be run on AC, DC, and solar for example.The entire instrument is compact and can be configured with any amountor numbers of reaction compartments necessary. The instrument can beportable to be used in the field (research for example) or carried to anarea of use. The number of reaction compartments typically would be10-20 per chamber and are stackable or are joined linearly or areconnected in any other manner which is appropriate. A portable fieldunit could have as few as 1-5, or 5-10, reaction compartments, forexample, for less weight. Preferably the components are made from lightweight, anti-corrosive materials. A further advantage of the presentinvention is that the instrument can be serviced in a modular approach.Each reaction compartment or slide support element in the module can beremoved in isolation and serviced or discarded and replaced with an allnew unit with simple modular attachments. All the motion controls arepreferably modular and either serviceable or completely replaceable. Anadvantage to this modular serviceability is that the other reactioncompartments that are in use or could be used, are not affected duringservicing of any device or part from a different reaction compartment.

An advantage of the present invention, as explained previously, is thateach slide can be treated with a separate unique reagent, inferring thatany slide can have any reagent and be treated at pressures and forvarying amounts of treatment times which are the same or different fromall other slides loaded into the apparatus. Examples of reagentsinclude, but are not limited to: antigen retrieval reagents, RNA and DNAprobes, citrate buffer, EDTA, TRIS, PBS, with or without surfactants ordetergents like SDS, Tween, Brij, ionic and non ionic detergents, andsilicone additives, rinse buffers, immunohistochemical reagents,histochemical reagents, in-situ hybridization reagents, PCR reagents,coverslipping reagents, silicone oils, mineral oils, detection reagentsand processing reagents, liquid reagents, reconstituted dry reagents,biological reagents and aqueous and non-aqueous reagents, anddeparaffinizing compositions of water with one or more siliconesurfactants or silicone additives. Another advantage with the presentinvention is that cross contamination from reagents or biologicalspecimens one slide by another slide is eliminated because each slide isseparated and treated with its own reagent in a separate reactioncompartment.

Another important advantage of present invention is that each individualreaction compartment can be cleaned or repaired separately andautomatically at the same time that other reaction compartments beingused to process slides. Thus, there is no downtime or interruption forthe other reaction compartments when a particular individual reactioncompartment is being cleaned or repaired. Each reaction compartment canbe separately cleaned and/or sterilized by steam, with or without adetergent or sterilizing reagent and dried with heated (below 100° C.)or super heated (above 100° C.) air. This type of sterilized cleaningcould be used for example if a biological specimen that was beingprocessed had infectious properties. Each reaction compartmentessentially has the properties of an individual self-regulated andcontrolled miniature autoclave. Sterilization of each reactioncompartment prior to use with the next biological specimen process canprovide an inherent technical advantage due to the elimination of crosscontamination and direct contact with infectious biological specimens.Sterilization can be performed using steam alone, or chemicals dispensedby a reagent strip or another dispensing element.

Reagent Strips

In a preferred embodiment of the invention, reagents are supplied to thereaction compartment from a reagent strip (also referred to herein as areagent dispensing strip) individualized for a single reactioncompartment as described in more detail below (FIGS. 1-22 and 39-78).

The reagent strip comprises at least one and preferably a plurality ofseparate reagent containers. The reagents in the reagent containers canbe of any type known in the art, including but not limited to, antigenretrieval reagents, RNA and DNA probes, citrate buffer, EDTA, TRIS, PBS,with or without surfactants or detergents like SDS, Tween, Brij, ionicand non ionic detergents, and silicone additives, rinse buffers,immunohistochemical reagents, histochemical reagents, in-situhybridization reagents, PCR reagents, coverslipping reagents, siliconeoils, mineral oils, detection reagents and processing reagents, liquidreagents, reconstituted dry reagents, biological reagents and aqueousand non-aqueous reagents, and deparaffinizing compositions of water withone or more silicone surfactants or silicone additives. The reagent canalso be a dry or desiccated reagent that can be dispensed onto abiological specimen or a dry or desiccated reagent that can bereconstituted prior to dispensing onto the biological specimen. A dry ordesiccated reagent can be dispensed onto the biological specimen andthen reconstituted by another reagent, for example. The reagent stripand individual reagent containers can be made out of any appropriatematerial, including, but not limited to, plastics, metals, polymers, andcomposites. The reagent strip, in a preferred version, has an openingbetween individual reagent containers which can be sealed by a reagentdispensing plunger on the apparatus to close an upper opening in thereaction compartment (described in more detail below). The reagent stripcan be constructed of materials which enable it to be heated, ifdesired, by a heating means located on or in the reagent strip and/orthe individual reagent containers, or heating means adjacent the reagentstrip and/or reagent container to pre-heat the reagents prior todispensing of the reagent from the reagent strip into the reactioncompartment and onto the microscope slides or onto the microscope slidebefore it is inserted into the reaction compartment. The heating meanscan be any known to those of ordinary skill in the art including, butnot limited to, infrared heating, electrical conductive inks, blanket,wire wrappings, light, kapton heaters, foil heater, and conductive typeheaters. The reagent strip, for example, can be directly wired to anelectrical supply for activating the heaters or can use wirelesstechnology to interface with the heaters located on the reagent strip orreagent containers.

The reagent strips in a preferred embodiment are 0.25 inch (6.35 mm) to3 inches (76.2 mm) in width and any length to accommodate a sufficientnumber of reagent dispensing containers to complete a staining protocol.The length in a preferred embodiment is from 4 inches (101.6 mm) to 20inches (508 mm) or greater, with a length of less than 10 inches (254mm) being preferred. The reagent strips can have any number, size,configuration, dispensing abilities (i.e., the ability to dispense areagent from the reagent strip under a pressure greater than that of thereaction compartment's internal pressure) of the reagent containers(i.e., capsules, blister packs, miniature syringe-type containers,dispensing (volume metered) containers, and volume metered dispensingcontainers that can be dispense their reagents at a pressure higher thanthe reaction compartment's internal pressures).

The reagent strips can have any number or configuration (arrangement orpositioning) of vapor vents, vapor holes, vapor releasing devices(pressure valve or pressure regulator), pressure monitoring devices, orcooling windows based on a particular protocol, for example, as shown inFIGS. 39-78, and as described and discussed below. Each reagent stripcan have reagent containers arranged thereon in a pattern such thatreagent from the first reagent container is dispensed at one end of thereagent strip and successive reagents are dispensed from successivereagent containers as the reagent strip moves forward toward the lastreagent container on the opposite end of the reagent strip withoutskipping or moving in an opposing direction. The reagent strip can alsobe utilized by dispensing reagents from non-successive reagentcontainers in an out-of-order arrangement on the reagent strip whereinthe reagent strip is moved “back and forth” to “pick” and “dispense”reagents from particular reagent containers. In a preferred embodimenteither method of dispensing (successive or non-successive) would be usedto dispense reagents from every reagent container present on the reagentstrip without leaving any one reagent unused.

Alternatively, the user can delete or override a pre-set protocol, withthe microprocessor, to skip any particular reagent container orcontainers on the reagent strip. The reagent strip can be placed on thereagent strip support device and is captured and fixedly held in placeon the support device by some means known in the art of securingdevices. An example of a securing device would be a “clip” such as usedon a “clipboard” to secure either end or either side of the reagentstrip to the reagent strip holder. Other securing devices which can beused are cogs, snaps, grabbers, low tack adhesives, “fitted” or “snug”fitting strip into the reagent strip holders rails, or other meansdescribed herein or known in the art.

Elevated pressure within the reaction compartment can cause reagentsplaced on the slide without or with heat to be pushed into closerphysical contact with the biological specimen on the slide, therebyimproving the staining. The pressurizing means (with or withoutadditional heating) can cycle on and off and mixing jets can be employedto mix the reagent and then repressurized to push the reagent down ontothe slide. This process can be repeated.

Reagents (e.g., gas and liquid reagents as described elsewhere herein)and all processing components, including, but not limited to, mixing airjets, vacuum (aspiration), pressure relief, air pressure, waste removal,and liquid reagents can be brought to the reaction compartment (inaddition to or in lieu of reagents supplied by a reagent strip) of eachreaction module (either into or outside of the reaction compartment) viapneumatic of electrically operated valves. These valves can be aseparate valve for each component being delivered to each reactionmodule for example by 2-port, multi-port, rotary type valves (multi-portvalve) and or pinch-type valves. The distribution of processingcomponents can be via one or more rotary valves per reaction module.Rotary valves can be used along with 2-port valves or pinch-valves inany combination with or without rotary valves. These miniature typevalves (e.g., multi-port valves, rotary valves, 2-port valves, and pinchvalves) are commercially available by vendors including Bio-Chem ValveCompany, 85 Fulton Street, Boonton, N.J. 07005, Parker Hannifan, 6035Parkland Boulevard, Cleveland Ohio 44124, and Tri-Tech LLC, 56733Magnetic Drive, Mishawaka, Ind. 46545.

Embodiments of FIGS. 1-22

Referring now to the drawings, shown in FIGS. 1-8 is a reconfigurablereagent dispensing strip (reagent strip) 10 which is constructed of abase 12, preferably constructed of a metal, plastic, thermoplastic orpolymeric material, and a plurality of reagent containers 14 (designatedfor ease of reference as A-H), each of which is positioned upon a tile16 of the base 12. The base 12 has an upper surface 18, a lower surface20, a first end 22 (near first reagent container A) and a second end 24(near last reagent container H). Each tile 16 preferably has a containerplatform 26 upon which each reagent container 14 is positioned upon, andis secured thereto via a container connector 30. Each tile 16 has aninjector aperture 28 which extends therethrough. Preferably between eachcontainer platform 26 is located a rinse port aperture 32 which extendsthrough the base 12. As shown in the figures, a separate rinse portaperture 32 is located between each container platform 26, but eachreagent strip 10 may have only a single or several rinse port apertures32. The reagent strip 10 preferably comprises a plurality of score lines34 (or perforation lines) individually located between pairs of tiles 16which enables the user to separate an individual tile 16 from the base12. Each tile 16 also has a plurality of tile connector receiving holes36 for enabling detached tiles 16 to be reconnected to an adjacent tile16. Each reagent container 14 comprises a body 40, an inner space 42within the body 40, a piston 44, an injector nozzle 46, and a reagent 48disposed in the inner space 42 between the piston 44 and the injectornozzle 46. In a preferred embodiment, the tiles 16 of the base 12 aremolded in a single piece and the reagent containers 14 are molded orpermanently attached to their respective tiles 16 in the base 12.

As shown in FIG. 4, an individual tile 16 can be separated along thescore line 34 from an adjacent tile 16, wherein, for example, the tiles16 having reagent containers G and H have been detached. Each tile 16and reagent container 14 constitutes a single reagent module 50 (e.g.,the reagent module 50 having reagent container G is referred to asreagent module 50G and the reagent module 50 having reagent container His referred to as reagent module 50H. FIG. 5 shows a reagent strip 10 ain which reagent module 50H has been reattached to reagent module 50Fvia a tile connector 52 (FIG. 6) which comprises two pairs of tileconnector link feet 54 and has an aperture which leaves the rinse portaperture 32 uncovered. In an alternative embodiment, shown in FIGS. 7and 8, a reagent strip 10 b is constructed of the original reagent strip10 except reagent module 50G has been replaced with a new reagent module50 gg positioned between reagent module 50F and reagent module 50H andconnected thereto via a pair of tile connectors 52.

The configurations of the reagent strips 10 a and 10 b are merelyexamples of how the configurations of reagent modules 50 can berearranged, as will be well understood by a person of ordinary skill inthe art.

Shown in FIGS. 9-16 is a reconfigurable reagent dispensing strip(reagent strip) 60 which is constructed of a base 62, preferablyconstructed of a metal, plastic, thermoplastic or polymeric material,and a plurality of reagent containers 78 (designated for ease ofreference as A-H), each of which is positioned upon a container platform72 of the base 62. The base 62 has an upper surface 64, a lower surface66, a first end 68 (near first reagent container A) and a second end 70(near last reagent container H). Each reagent container 78 is secured tothe base 62 via a container female connector 74. Each container platform72 has an injector hole 82 which extends through the base 62. Preferablybetween each container platform 72 is located a rinse port aperture 76which extends through the base 62. As shown in the figures, a separaterinse port aperture 76 is located between each container platform 72,but each reagent strip 60 may have only a single or several rinse portapertures 76. Each reagent container 78 comprises an injector 80 and acontainer male connector 84 which is insertable into a correspondingfemale connector 74 for securing the container 78 to the containerplatform 72. The container female connector 74 and container maleconnector 84 may comprise a twist lock. Each reagent container 78 isotherwise constructed in a manner similar to container 14 of reagentstrip 10. In a preferred embodiment, the base 62 is molded in a singlepiece and the reagent containers 78 are attachable and removable fromtheir respective container platforms 72 in the base 62.

As shown in FIGS. 11 and 13, an individual reagent container 78 can beremoved, for example, reagent containers 78 designated as G and H havebeen detached. FIG. 15 shows a reagent strip 60 a in which reagentmodule 78H has been reattached to the container platform 72 whichoriginal held reagent container 78G. In an alternative embodiment, shownin FIG. 16, a reagent strip 60 b is constructed of the original reagentstrip 60 except reagent container 78G has been replaced with a newreagent container 78 GG positioned between reagent module 78F andreagent container 78H and connected thereto upon container platform 72G.

The configurations of the reagent strips 60 a and 60 b are merelyexamples of how the configurations of reagent containers 78 can berearranged, as will be well understood by a person of ordinary skill inthe art.

Shown in FIGS. 17-22 is a reconfigurable reagent dispensing strip(reagent strip) 90 which is constructed of a plurality of interlockingreagent modules 92 each comprising a reagent container 94 (designatedfor ease of reference as A-H), and an interlocking tile 96. The reagentstrip 90 has an upper surface 104 and a lower surface 106. Eachinterlocking tile 96 has an injector aperture 97 which extendstherethrough. Preferably between each reagent container 94 is located arinse port aperture 102. As shown in the figures, a separate rinse portaperture 102 is located between each reagent container 94, but eachreagent strip 90 may have only a single or several rinse port apertures102. Each interlocking tile 96 of the reagent strip has a jigsaw-likemale interlocking portion 98 and jigsaw-like female interlocking portion100, each of which is connectable to an adjacent female interlockingportion 100 and a male interlocking portion 98, respectively. Thisenables the user to separate and reattach individual interlocking tiles96. Each reagent container 94 is constructed in a manner similar to thatof reagent container 14. In a preferred embodiment, each interlockingtiles 96 is molded in a single piece with the reagent container 94 or ispermanently attached in any manner known in the art.

As shown in FIG. 17-19, individual interlocking tiles 96 can beseparated from adjacent interlocking tiles 96, wherein, for example, theinterlocking tiles 96 having reagent containers G and H have beendetached. Each interlocking tile 96 and reagent container 94 constitutesa single interlocking reagent module 92 (e.g., the interlocking reagentmodule 92 having reagent container G is referred to as reagent module92G and the reagent module 92 having reagent container H is referred toas reagent module 92H. FIG. 21 shows a reagent strip 90 a in whichreagent module 92H has been reattached to reagent module 92F via maleinterlocking portion 98 and female interlocking portion 100 and has arinse port aperture 102. In an alternative embodiment, shown in FIG. 22,a reagent strip 90 b is constructed of the original reagent strip 90except reagent module 92G has been replaced with a new reagent module92GG positioned between reagent module 92H and reagent module 92F andconnected thereto.

The configurations of the reagent strips 90 a and 90 b are merelyexamples of how the configurations of interlocking reagent modules 92can be rearranged, as will be well understood by a person of ordinaryskill in the art.

As described above and elsewhere herein, in a preferred embodiment, thereagent strip of the present invention can be reconfigured from anoriginal or previous configuration, by the user, thereby giving the userthe ability to custom arrange the reagent containers 14 on the reagentstrip 10, e.g., as shown for reagent strips 10 a and 10 b.

For example, if a user does not want or need to use all the reagentcontainers 14 present on a preassembled reagent strip 10, or the userwould like to add one or more reagent containers 14 to a particularreagent strip or rearrange them thereon, the user will have the abilityto reconfigure the reagent containers 14 on the reagent strip.

As shown in FIGS. 1-4, in one embodiment, the preassembled tiles 16having reagent containers 14 positioned thereon on the reagent strip 10can be perforated or easily separated via tile score lines 34 to producereagent modules 50 thereby enabling the reconfiguration of the reagentmodules 50 as described elsewhere herein. The reagent tiles 16 can beany size but preferably those in the same reagent strip 10-10 b are allthe same size, although certain reagent strips may have tiles 16 thatare different in size.

For example, in a reagent strip of the present invention such as reagentstrip 10 which is 5 inches long and has 10 individual tiles 16 present,each tile 16 would be 0.5 inch in length. In the embodiment of FIG. 1,the separate tiles 16 are manufactured together in a series and areseparable via tile score lines 34 such as grooves, perforations or othermeans of detaching between the individual tiles 16 as discussedelsewhere herein.

As indicated above, a reagent strip 10 has four tile connector receivingholes 36 in each tile 16. Adjacent tiles 16 can be joined and held by atile connector 52 similar to a master bicycle chain link. There are manyways known by those of ordinary skill in the art of how to separate andjoin small parts which could be used to link the tiles 16 together in away to produce the reconfigured reagent strips of the present invention.

The “snap together” or “puzzle piece” approach if reagent strips 90-90 bmakes the reagent strip continuous wherein it can be pulled or pushedover the reaction compartment of the apparatus without dislodging one ofthe reagent tiles and without requiring a separate attaching means tohold the strip together (e.g., a connector).

Any of the reagent strips contemplated herein can include any or all thefeatures noted above, and may also have empty reagent containers 14which can be filled by the user. In fact, in one embodiment, the reagentstrip of the present invention may comprise empty containers 14 for theuser to “custom make” his own custom reagent strip using joined emptyreagent modules and/or separate tiles to build a reagent strip.

In one embodiment, the reagent strip of the present invention may have apredetermined number of reagent containers in a predetermined sequencethat cannot be altered by the user (e.g., see FIGS. 39-78 as describedbelow in more detail). The reagent strip is thus non-reconfigurable. Ina preferred embodiment the non-reconfigurable reagent strip is advancedin one direction intermittently as directed by the microprocessor orother means until the all of the reagents in the reagent strip have beendeployed. The reagent strip is preferably disposable and is in apreferred embodiment is thrown away and not refilled or reused again.Each reagent container can be deployed consecutively, or one or morecontainers could be skipped over, then returned to. The reagent stripcould be labeled with a computer readable optical character symbol orcode (bar code, optically readable symbol, code, character) to identifythe type of treatment protocol which would then program the computer asto the type of protocol to be used, and when the next reagent in theseries would be dispensed onto the slide.

Once the microprocessor, along with the optical reader on theinstrument, has scanned the optical character on the reagent strip, theuser would then place the reagent strip on the reagent strip supportdevice and press the start button located near the opening of theindividual reaction compartment or on the corresponding icon on thecomputer screen to start the procedure. The reagent strip support devicewould preferably have a homing device so the computer would “know” werethe first reagent container is located relative to the dispensingplunger. In a preferred embodiment, the distance between reagentcontainers on the reagent strip would be an equal distance (e.g., 0.5inches apart) so once the homing position is recognized by themicroprocessor, it will know where the first reagent container is on thereagent strip and the reagent strip will be moved to the reagentdispenser position where the reagent will be dispensed onto thebiological specimen on the microscope slide. The reagent strip will thenbe moved 0.5 inches (or other predetermined equal distance) to the nextreagent container (or to a rinsing port there between) after theappropriate amount of time. The protocol type, treatment times perreagent, rinse steps, drying, air mixing, etc., are all assessed at thetime the optical character is scanned and recognized by themicroprocessor.

In this embodiment, it is preferably predetermined that all the reagentson the reagent strip will have to be dispensed onto the slide within agiven duration of time and processing conditions. The microprocessorwill move and activate all the processing devices independently for allreaction compartments until the last step of the protocol is completed.

The distance between reagent containers on the reagent strip can be anydistance that the microprocessor could identify (e.g., from 0.001 inchto about 4.00 inches or greater). The identification of an individualreagent container by the microprocessor can be by consistent distancesafter initial homing, each reagent container optionally having its ownseparate homing device on the reagent strip or on the reagent stripsupport device, optical recognition, and any other type known in the artfor recognizing multiple containers by a microprocessor.

In a further embodiment, the reagent strip or containers or reagentmodules thereof could have numeric symbols (numbers) or other symbols(characters) printed thereon that the user simply inputs into themicroprocessor for identification of the reagent containers of thereagent strip and protocols associated therewith.

In a preferred embodiment, the reagent strip of the present invention isa single use device which is disposed of after its use, wherein thesingle use reagent strip is completely or partially prepared by the user(e.g., the user fills one or more containers for the reagent strip) oris completely prepared by a manufacturer. Alternately, the reagent stripcould be reusable wherein individual containers could be refilled by auser or manufacturer or new containers or reagent modules could be addedto a used reagent strip or could be substituted for used containers orreagent modules on a used reagent strip. Further, a single usedcontainer could still have several “doses” or “applications” of reagentwherein it would be advantageous for the user to switch the usedcontainer from a used reagent strip to a different reagent strip.Further, as described elsewhere herein, the reagent strip could bereconfigurable such that one or more containers or reagent modules couldbe replaced, substituted, rearranged or “switched-out” for an alternateone or more containers.

Embodiments of FIGS. 23-38B

Shown in FIGS. 23-38B is a reaction module 110 having a cylindricalreaction compartment 112, a slide support element 114, and a reagentstrip support device 116, as previously described. Preferably, thereaction compartment 112 has an inner diameter of 2-5 cm, and morepreferably 27 mm, and has a wall thickness of 2 mm to 3 cm. The lengthof the slide support element 114 is preferably 10-20 cm, and morepreferably 12 cm. The length of the reaction compartment 112 ispreferably 15-30 cm, and more preferably 20 cm. The reagent stripsupport device 116 is operatingly connected (e.g., attached at a top) tothe reaction compartment 112 via a reagent conduit 122 which opens tothe inner space 120 of the reaction compartment 112. There is aninjector port orifice 124 in the reagent strip support device 116 whichis adapted to receive the injector nozzle from a reagent container of areagent strip. The reagent strip support device 116 has a front end 126and a rear end 128. The reagent strip support device 116 functions toreceive, support, and eject a reagent strip of the present invention.The slide support element 114 has a base 134 which can reciprocatinglybe moved into and out of the reaction compartment 112. The slide supportelement 114 comprises a heating element 136 upon which a slide 140 isplaced. The slide support element 114 may have a handle 142 whichenables a technician to more easily insert and withdraw the base 134from the reaction compartment 112. The slide support element 114preferably further comprises a sealing means which in the presentembodiment is a front O-ring 144 and a rear O-ring 145 for providing apressure resistant seal of the base 134 against the inner surface 118 ofthe reaction compartment 112. The slide support element can beconstructed from materials which include, but are not limited to, glass,quartz, Pyrex®, borosilicate, steel, metals, aluminum, composites,polymers such as polycarbonate and plastics or combinations thereof.

The slide support element 114 also preferably has a drainage port 146for receiving and draining reagents and waste liquids from the reactioncompartment 112. The slide support element 114 further preferably hasone or more cooling ducts 148 which are operatively connected to a subheating element cooling space 148 a beneath the heating element 136, andone or more cooling duct exits 148 b which evacuate the cooling air orliquid from the sub heating element cooling space 148 a. The slidesupport element 114 preferably further comprises a first air/pressureduct 150 and a second air/pressure duct 152 for regulation of thepressure within the reaction compartment 112 as discussed elsewhereherein. The duct 150 and/or duct 152 or an additional duct (not shown)can be used for releasing and/or regulating pressure from the reactioncompartment 112. The slide support element 114, as noted above,comprises a heating element 136 upon which the microscope slide 140 isplaced for application of reagents thereon. The reaction module 110 mayfurther comprise a thermocouple or other temperature measuring devicefor measuring temperatures of the slide or other components therein.Before operation the slide support element 114 is inserted by a slidingmotion into the inner space 120 of the reaction compartment 112 (seeFIG. 24A). Also before operation the reagent strip 10 (or any otherreagent strip described or enabled herein) is inserted into the reagentstrip support device 116, for example, inserting the first end 22 of thereagent strip 10 into the front end of 126 of the reagent strip supportdevice 116, wherein during operation the reagent strip 10 is moved in adirection toward the rear end 128 of the reagent strip support device116. The reagent strip 10 may be advanced manually or automatically viaa pulling or pushing device, including rollers or a track whichincrementally advances the reagent strip 10 as instructed by themicroprocessor. The reaction module 110 further comprises a reagentconduit 122 in the reaction compartment 112 for allowing passage of areagent from the reagent strip 10 into the reaction compartment 112. Thereaction module 110 also comprises a dispenser plunger 154 (alsoreferred to herein as a dispensing element), which has a dispensingcanal 156 therein for allowing passage of another reagent or solutiontherethrough preferably from a remote source. The reagent strip supportdevice 116 preferably has an injector port orifice 124 for receiving atleast a portion of an injector nozzle 46 from a reagent container 14 ofthe reagent strip 10 during use thereof.

The slide support element 214 may further optionally comprise one ormore drainage and/or supply conduits which lead to the base cavity 252for supplying the base cavity 252 with a liquid or other solution andfor draining used liquid from the base cavity 252 after its use (e.g.,by aspiration). Other supply ports, conduits, and ducts may supply thereaction compartments of the present invention as described previouslyin U.S. Pat. No. 6,534,008 and U.S. Pat. No. 6,855,292.

During operation, as shown in FIGS. 24A-24B and 29, a reagent strip 10(or any other reagent strip described or enabled elsewhere herein) isinserted into the reagent strip support device 116 as previouslydescribed and a reagent container 14 is positioned over the injectorport orifice 124. The dispensing plunger 154 is extended downwardly intothe inner space 42 of the reagent container 14 wherein it engages thepiston 44, forcing the piston 44 downwardly and causing ejection of thereagent 48 through the injector nozzle 46, through the reagent conduit122 and providing reagent 158 deposited onto the slide 140. When thedispensing plunger 154 forces the piston 44 downwardly, a seal ismaintained within the reagent container 14 and in a preferred embodimentenables pressurization of the reaction compartment 112. The reagent 158can be mixed on the microscope slide 140 by delivering bursts of air 162through the first air/pressure duct 150 and the second air/pressure duct152 as discussed in further detail below. In a subsequent step thedispensing plunger 154 may be withdrawn (FIG. 25A-B) and the base 134 ofthe slide support element 114 tilted within the reaction compartment 112to allow the reagent to drain from the slide 140, forming a reagentdrainage 160 which is collected in the drainage port 146, removed fromthe reaction compartment 112, and collected in a waste storage container(not shown). In a later step (FIGS. 26A-B) the slide 140 is returned toan upright, horizontal position and the reagent strip 10 is advanceduntil the rinse port aperture 32 is positioned above the injector portorifice 124 wherein rinse solution 163 is delivered from a rinsesolution reservoir (not shown). Furthermore, air or liquid may bedelivered through the dispensing canal 156 in the dispensing plunger 154to cause mixing of reagent 158 or to remove the reagent 158 from theslide, or to enhance the rinsing of the reagent 158 or rinse solution163 from the slide 140 (e.g., see FIGS. 27A-B). Finally as shown in FIG.28, after all reagents from the reagent strip 10 have been dispensed,the portion of the slide support element 114 which carries the slide 140is withdrawn from the reaction compartment 112 wherein the slide 140 isthen removed from the slide support element 114. Note that FIGS. 29-30are enlarged versions of FIGS. 24A and 26A, respectively and areprovided herein for the purpose of more easily showing the stepstherein.

FIGS. 31A-32B provide a more detailed description of how the bursts ofair 162 delivered form the first air/pressure duct 150 and secondair/pressure duct 152 can be used to cause mixing of the reagent 158 onthe slide 140. Preferably, the first air/pressure duct 150 and secondair/pressure duct 152 are operated alternately to provide bursts of air162 in alternating clockwise/counterclockwise directions to agitate thereagent 158. The first air/pressure duct 150 and second air/pressureduct 152 can be used simultaneously to pressurize the reactioncompartment 112. At any desired time the heating element 164 can be usedto heat the slide 140 and reagent 158 thereon as discussed in greaterdetail elsewhere herein. As shown in FIGS. 33-35B after the slide 140 isheated, it can be rapidly cooled by directing air or liquid via thecooling ducts 148 into sub heating element cooling spaces 148 a whichare located below the heating element 164 which in one embodiment islocated below and is used to heat a hot plate 166 upon which the slide140 is positioned. Air or liquid used for cooling can then pass throughcooling duct exits 148 b. In another embodiment, shown in FIGS. 36-38B asub heating element cooling space 148 c is similar to sub heatingelement cooling space 148 a except the cooling air or liquid whichpasses through the sub heating element cooling space 148 c is deliveredvia one of the cooling ducts 148 and exits the slide support element 114via the outer cooling duct 148.

Other embodiments of reagent strips of FIGS. 23-38B which have featuressimilar to those of FIGS. 39-78, having various combinations of ventholes, ventilation slots, and rapid cooling windows can readily beenvisioned particularly regarding the sizes, shapes and locations of thevent holes, ventilation slots and rapid cooling windows.

Each individual reagent containers of the present invention, e.g.,reagent containers 14, 78 and 94, can be a container whose inner spacehas been evacuated to hold a vacuum. To fill the reagent container, areagent source can be contacted with a filling port of the reagentcontainer (not shown) wherein the vacuum then pulls the reagent into theinner space of the reagent container. The reagent container volumelimits the amount of reagent pulled into the reagent container from thereagent source.

Alternatively, the reagent container may have a plunger or pistonpresent in a down (dispense) position. The dispensing port (e.g., theinjector nozzle) of the reagent container is connected to a reagentsource. Reagent is drawn into the reagent container by moving theplunger or piston in the reagent container upwardly. Once the plunger orpiston has reached its uppermost position, the individual reagentcontainer is filled. The filling of a reagent container could be assimple as a method of filling a common syringe with a reagent andaffixing the outlet of the syringe to the bottom of the individualreagent container and pushing the reagent into the reagent containerthus moving the reagent container's plunger or piston upwardly andfilling the reagent container. The bottom of the injector nozzle of eachreagent container would then be sealed or capped with its own individualcap or seal. The plurality of caps or seals on a reagent strip may beremovable together. This linkage is useful in one embodiment to removethe caps in one motion to expose the injector nozzles prior to puttingthe reagent strip on the reagent strip support device. In an alternateembodiment of the sealing of the injector nozzles (i.e., the dispensingside of the reagent strip) a cover made of foil, plastic, or othercovering means can be used that can be peeled away prior to use toexpose the injector nozzles.

In a preferred embodiment, the injector nozzles, reagent strips, and thedispensing plunger or piston or other ducts leading to the reactionmodule can dispense reagents by using a pressure which is greater thanthe internal pressure of the reaction compartment into which the reagentis dispensed. For example, if a reaction compartment is pressurized at30 psig (308.1 kPa), a reagent must be dispensed into the reactioncompartment with force exceeding 30 psig (308.1 kPa) to overcome thepressure in the reaction compartment. Otherwise, the reactioncompartment would have to be depressurized to add the reagent. Such adepressurization step would probably be deleterious because thedepressurization would cause the reagent on the slide to boil off due toextreme evaporation at high temperature. The present invention candispense its reagents under pressure in the range of over 0 to 350 psig(101.3 kPa-2514 kPa), preferably in the range of 0.5-100 psig (104.8kPa-790.6 kPa) and more preferably 5-50 psig (135.8 kPa-446.0 kPa) andthe reaction compartment can be pressurized to these levels as well.

The reagent strips of the present invention are used to provide reagentsonto microscope slides positioned in, or prior to being positioned in, apressurizable reaction compartment of the antigen retrievable apparatusof the present invention as shown for example in FIGS. 23-38B.

As shown in FIGS. 23-38B, each reaction compartment of the apparatuspreferably comprises a hollow cylinder, preferably constructed of athermoplastic resin or polymer (including but not limited topolycarbonate or any other polymeric material able to withstand elevatedtemperatures and pressures), glass, Pyrex®, quartz, other crystallinematerials, and metals and metal alloys. The tubular nature of thereaction compartment is preferred because the elevated pressures createdwithin the reaction compartment during its use are more evenlydistributed therein.

The seal between the slide support element and the reaction compartmentcan be formed using O-rings, as shown in the FIGS. 23-38B or can beformed using an inflatable O-ring, a seal, or an inflatable sealdepending on the shape of the mating surfaces. The seal can beconstructed of plastic, polymer, thermoplastic, resin, ceramic, rubber,metal glass, or composite, for example. In a preferred embodiment, themating surfaces of the slide support element and the reactioncompartment are of a low tolerance ground or polished sealing surface.These sealing surfaces when joined together eliminate the need for avisually or seal raised above the mating surface. In this embodiment,the ground or polished mating surface alone, when joined together,produces a microscopic seal with a large surface area to seal thereaction compartment and maintain an elevated pressure therein (aboveatmospheric) even under high temperature conditions above 100 degreescentigrade. The material of the slide support element and the tubularreaction compartment can feature a very high tolerance ground orpolished seal on the mating surfaces. In the preferred embodiment, theslide support element and the reaction compartment is made of a hightempered glass material like Pyrex®, or any material that can produce aground or polished mating surface to form a seal which maintains apressure above atmosphere pressure. The ground glass surface, orpolished surface of the slide support element against the ground orpolished surface of the reaction compartment yields an air-tight andpressure-tight seal when the two ground or polished surfaces are joinedtogether, wherein, there is no separate replaceable or raised seal tofill the mating surfaces void. This embodiment of the present inventioneliminates the need for raised seals (e.g., O-rings) thus reducingmaintenance cost for the replacement of separate components seal such asO-rings and increases simplicity and efficiency and seals the reactioncompartment even under pressures above atmospheric levels (e.g., above14.7 psig (101.325 kPa), i.e., above 0 psig (101.325 kPa)) and hightemperature conditions above 100° C. degrees centigrade.

The apparatus of the present invention preferably comprises a pluralityof reaction modules, such as the reaction module 110 shown in FIG. 23.Each reaction module 110 comprises a tubular reaction compartment 112, aslide support element 114 and a reagent strip support device 116. Thereaction compartment has an inner surface 118 and an inner space 120into which the slide support element 114 can be moved for treating abiological sample on a microscope slide 140 thereon. The slide supportelement 114 is able to slide into and out of the reaction compartment112 in a manner similar to a piston within a cylinder. When the slidesupport element 114 is withdrawn from the reaction compartment 112, aslide 140 can be placed thereon or removed therefrom. The slide supportelement 114 can be inserted into the reaction compartment 112 fortreatment of the material on the slide 140 as described elsewhereherein. As shown below, the slide support element 114 can be turned(tipped) within the reaction compartment 112 for facilitating theremoval of reagents or fluids from the slide 140 after the slide 140 hasbeen treated, as shown in the figures (e.g., see FIG. 25B). Reagents orfluids on the slide 140 can be mixed by air circulation as shown inFIGS. 31A-32B for example. After heating, the slide 140 can be cooled bycirculation of air or fluid thereunder, for example as shown in FIGS.34A-38B. In another embodiment, the slide 140 could be cooled by using acirculating liquid such as a reagent that becomes pre-heated by passingunder the heated slide 140 thus transferring heat to the circulatingreagent which could then be dispensed onto the slide 140.

The reaction compartment 112 can be constructed of any material known inart of high temperature and pressure compatible devices. These materialsalso include, but are not limited to, plastic, composites, ceramics,metals and coated metals. The instrument can be coated for resistance toporosity, to increase hydrophobic and hydrophilic properties, for easeof cleaning, chemical resistance, and stain resistance. These coatingscould be, for example, Teflon®, fluoropolymers, any other known coatingthat would impart these desirable properties to all surfaces of reactioncompartment 112 and surrounding structures with a different coatingbeing present on different portions of the apparatus. In one embodiment,for example, the inner surface 118 of the reaction compartment 112 iscoated with a hydrophobic, chemical, and stain resistant coating to aidin the draining of the condensed reagents on the inner surface 118 ofthe reaction compartment 112 and ease of removal of reagents therefrom.

The slide support element 114 of the reaction module 110 preferablycomprises a heating element 136, and a hot plate (which may be one andthe same) and which may include guide clips 138 or pegs or elements tolocate and secure the slide 140 thereon. The tops of the clips 138 maybe positioned to be below an upper surface of the microscope slide 140,so as to prevent reagent on the slide 140 from being wicked off by theclips 138 by capillary action.

In a particularly preferred embodiment, underneath the heating element136 is one or more recessions (sub-heating element cooling spaces 148 a)which are connected via cooling ducts 148 to a gas or liquid supplysource to quickly cool the heating element 136 thereby quickly coolingthe microscope slide 140 and the reagent thereon.

The slide support element 114 and reaction compartment 112 can beconstructed of any material suitable for use under pressurizedconditions and resistant to corrosion by laboratory reagents, includingbut not limited to stainless steel, metals, plastics (clear or opaque),polymers (e.g., polycarbonate), tempered glass, and Pyrex®.

Containment of waste and used reagents from the reaction module 110 willbe now briefly discussed, and will be discussed in more detail below.

In a preferred embodiment the apparatus of the present invention has awaste container (not shown) which can be connected to all the reactionmodules 110 by a fitting that can join multiple tubes or conduits. In apreferred embodiment of the present invention, this main fitting (notshown) can be joined to the waste container (which preferably isdisposable or non-reusable) by a breakable joint present on the wastecontainer. This fitting on the waste container snaps together with themain fitting of the instrument. This attachment is secure and will notleak under pressure. When detached, this fitting on the waste containerpartially “breaks away” and leaves behind on the waste container anairtight, leakproof, tamper proof, non-removable seal. The residualpiece that was detached from the waste container is removed by thetechnician and then is ready to be reattached to a new waste container.The waste container is now ready to be deposited in its entirety bymedical waste personnel. No other sealing is necessary. The tamper proofseal of the separated fitting protects the medical waste personnel fromcoming in contact with any of the waste in the sealed waste container.

In an alternate embodiment the detachable fitting on the waste containermay not have any residual piece on the main instrument fitting butrather “breaks” or “snaps” away form the detachable piece on thedisposable waste container cleanly.

In an alternate embodiment the instrument could have two or more wastecontainers wherein it is possible to remove one full waste containerwhile retaining one or more other waste containers attached to receivewaste from the working reaction modules. The microprocessor could alertthe technician that a waste container is in need of replacing by asensor located in the waste container. If the technician chooses toignore the alert from the instrument, it could divert the waste toanother waste container until the time is convenient to replace the fullwaste container. Since the processing device operates each reactionmodule 110 independently, the waste containers are set-up to receivewaste from any of the working reaction modules 110 eliminating the needto stop the instrument to change any full waste container. The wastecontainers can be hooked up in a series or in parallel to keep at leastone waste container active while any other waste container is beingchanged. The microprocessor is in direct communication with all thewaste containers and will shut down waste routes that are going to afitting that has been detached and is in the process of replacement.

In an alternate embodiment, the instrument could have one main wastecontainer which when full would alert the technician to start the wasterecovery procedure. The main waste container could be drained to asecondary waste container to be disposed.

The waste container can be charged with activated charcoal or otherneutralizing chemicals to aid in decontamination. The waste containercan have a vent that has a neutralizing filter to release the buildup ofpressured vapors.

Turning again to the figures, it will be shown in greater detail how thereaction module 110 (and others described herein) operates.

As explained above, the operation sequence of the reagent strip 10 withthe reaction module 110 is generally shown in FIGS. 23-38B.

The slide 140 is loaded onto the heating element 136 or the hot plate166 of the slide support element 114 and positioned by location clips138 or guide pegs or other orientation elements to verify properlocation of the microscope slide 140 on the slide support element 114.The slide support element 114 and slide 140 is then moved into thereaction compartment 112 wherein it is sealed via the O-rings 144 and145. The reagent strip 10 is placed onto the reagent dispensing stripsupport device 116. The protocol is entered either automatically ormanually (described elsewhere herein) and the instrument with theplurality of reaction modules 110 is instructed to start. Depending onthe protocol the heating element 136 can start to heat the slide 140 orthe protocol instructs the dispensing of a reagent from the reagentstrip 10 or from another source via the dispensing plunger 154.

If an individual reagent container 14 located on the reagent strip 10 isselected, that particular reagent container 14 will be positioned overthe injector port orifice 124, and the dispensing plunger 154 anddepresses the piston 44 within the reagent container 14 to expel thereagent 48 therefrom onto the microscope slide 140. The reagent strip 10would then be moved to position the rinse port aperture 76 in thereagent strip 10 (e.g., generally located between adjacent reagentcontainers 14) over the injector port orifice 124 wherein the dispensingplunger 154 would be lowered to seal the injector port orifice 124 or,additional air or reagent could be injected into the reactioncompartment 112. Once the reagent 158 which has been applied to theslide 140 is removed from the slide 140 by tilting the slide 140 or byrinsing, the slide 140 can be further rinsed with a reagents or treatedwith pressurized air from the dispensing plunger 154.

As disclosed in U.S. Pat. Nos. 6,534,008 and 6,855,292, the apparatus ofthe present invention used to treat the microscope slide comprises aplurality of reaction modules 110 each having a reaction compartment 112which is encloseable for reducing evaporative heat loss by vapors beingcontained inside the reaction compartment 112 during heating conditions.

As discussed elsewhere herein, the reaction compartments of the reactionmodules of the present invention can be pressurized (positively ornegatively) during heating of the reaction compartment or pressurizedwithout heating, or pre-pressurized (positively or negatively) beforethe microscope slide or other component of the reaction module isheated. The reaction compartment can be pre-pressurized, then heated,then repressurized to maintain a preferred pressure level within thereaction compartment. The reaction compartment can be pressurized eitherby vapor, gas, or steam produced by a reagent, solution, or liquidwithin the reaction compartment or by air, steam, inert gases, N₂ or anyother gas typically used for pressurizing vessels, which is providedfrom an external source and is supplied via air/pressure ducts orconduits or vacuum lines into the reaction compartment.

Embodiments of FIGS. 39-78

Various embodiments of reagent strips which can be used with the presentinvention, and which may also be used in the apparatus of U.S. Pat. Nos.6,534,008 and 6,855,292, are shown in FIGS. 39-78. These reagent stripshave various holes, openings, slits and windows for controlling theventing of vapors produced within the reaction compartments 112, asdescribed below.

Alternate embodiments of reagent strips which may be used in the presentinvention are shown in FIGS. 39-78 herein and are similar to embodimentsof reagent strips used and described in U.S. Pat. Nos. 6,534,008 and6,855,292, and U.S. Ser. Nos. 10/245,035 and 10/943,386.

Shown in FIGS. 39-42 is a reagent strip 170 which can be used in analternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 has a base 172, an upper surface 171, a lower surface173, a plurality of reagent capsules 174, each having a reagentdispensing port 176 thereunder, and a plurality of vent holes 178positioned between a pair of the reagent capsules 174. The vent holes178 allow excess vapor to escape from the reaction compartment therebypreventing excessive pressure buildup within the reaction compartment.

Shown in FIGS. 43-46 is another reagent strip 170 a which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 a has a base 172 a, an upper surface 171 a, a lowersurface 173 a, a plurality of reagent capsules 174 a, each having areagent dispensing port 176 a thereunder, and a plurality of vent holes178 a positioned between adjacent one of the reagent capsules 174 a. Thevent holes 178 a allow excess vapor to escape from the reactioncompartment thereby preventing excessive pressure buildup within thereaction compartment.

Shown in FIGS. 47-50 is another reagent strip 170 b which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 b has a base 172 b, an upper surface 171 b, a lowersurface 173 b, a plurality of reagent capsules 174 b, each having areagent dispensing port 176 b thereunder, and a plurality of vent holes178 b positioned between near an end of the reagent strip 170 b. Thevent holes 178 b allow excess vapor to escape from the reactioncompartment thereby preventing excessive pressure buildup within thereaction compartment.

Shown in FIGS. 51-54 is another reagent strip 170 c which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 c has a base 172 c, an upper surface 171 c, a lowersurface 173 c, a plurality of reagent capsules 174 c, each having areagent dispensing port 176 c thereunder, and a ventilation slot 180 cpositioned between a pair of the reagent capsules 174 c. The ventilationslot 180 c allows excess vapor to escape from the reaction compartmentthereby preventing excessive pressure buildup within the reactioncompartment.

Shown in FIGS. 55-58 is another reagent strip 170 d which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 d has a base 172 d, an upper surface 171 d, a lowersurface 173 d, a plurality of reagent capsules 174 d, each having areagent dispensing port 176 d thereunder, a ventilation slot 180 d and arapid cooling window 182 d positioned between a pair of the reagentcapsules 174 d. The ventilation slot 180 d allows excess vapor to escapefrom the reaction compartment thereby preventing excessive pressurebuildup within the reaction compartment, and the rapid cooling window182 d increasing the speed at which the microscope slide, and reactioncompartment cool down after a heating step.

Shown in FIGS. 59-62 is another reagent strip 170 e which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 e has a base 172 e, an upper surface 171 e, a lowersurface 173 e, a plurality of reagent capsules 174 e, each having areagent dispensing port 176 e thereunder, and a ventilation slot 180 eand a rapid cooling window 182 e. The ventilation slot 180 e allowsexcess vapor to escape from the reaction compartment thereby preventingexcessive pressure buildup within the reaction compartment, and therapid cooling window 182 e increases the rate at which the microscopeslide and reaction compartment cool down after a heating step.

Shown in FIGS. 63-66 is another reagent strip 170 f which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 f has a base 172 f, an upper surface 171 f, a lowersurface 173 f, a plurality of reagent capsules 174 f, each having areagent dispensing port 176 f thereunder, and a ventilation slot 180 fpositioned in the center of the base 172 f between adjacent one of thereagent capsules 174 f. The ventilation slot 180 f allows excess vaporto escape from the reaction compartment thereby preventing excessivepressure buildup within the reaction compartment.

Shown in FIGS. 67-70 is another reagent strip 170 g which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 g has a base 172 g, an upper surface 171 g, a lowersurface 173 g, a plurality of reagent capsules 174 g, each having areagent dispensing port 176 g thereunder, and a plurality of vent holes178 g positioned adjacent a pair of the reagent capsules 174 g. The ventholes 178 g allow excess vapor to escape from the reaction compartmentthereby preventing excessive pressure buildup within the reactioncompartment. The reagent strip 170 g further comprises a rapid coolingwindow 182 g to accelerate cooling of the microscope slide and thereaction compartment.

Shown in FIGS. 71-74 is another reagent strip 170 h which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 h has a base 172 h, an upper surface 171 h, a lowersurface 173 h, a plurality of reagent capsules 174 h, each having areagent dispensing port 176 h thereunder, a ventilation slot 180 h, anda rapid cooling window 182 h. The ventilation slot 180 h allows excessvapor to escape from the reaction compartment thereby preventingexcessive pressure buildup within the reaction compartment and the rapidcooling window 182 h increases the rate at which the microscope slideand reaction compartment cool down after a heating step.

Shown in FIGS. 75-78 is another reagent strip 170 i which can be used inan alternate embodiment of the presently described apparatus which has adispensing plunger which can crush a capsule containing a reagent.Reagent strip 170 i has a base 172 i, an upper surface 171 i, a lowersurface 173 i, a plurality of reagent capsules 174 i, each having areagent dispensing port 176 i thereunder, a plurality of vent holes 178i, and a rapid cooling window 182 i. The vent holes 178 i allow excessvapor to escape from the reaction compartment thereby preventingexcessive pressure buildup within the reaction compartment, and therapid cooling window 182 i increases the rate at which the microscopeslide and reaction compartment cool down after a heating step.

The vent structures of the reagent strips of FIGS. 39-78 are designed toallow sufficient heat to be contained within the reaction compartment112 by controlling the amount of vapor loss from the reagent to produceand maintain boiling conditions of reagents. It is known that aqueousreagents will boil at different temperatures in relation to the amountof solute contained in the solution. It is also known that when boilingsmall amounts of aqueous liquids (e.g., 500 microliters to 100 ml) thesolution will reach a particular boiling point at which its liquid phasewill go to its gaseous phase. This progression should to be controlledto reduce the amount of evaporative heat loss from the reactioncompartment.

An example of why the ability to control the release of evaporativevapors is important to maintaining boiling conditions is demonstrated byplacing an open-top chamber about a slide. During heating to boilingconditions small gaseous bubbles will form at the surface of the slidewhere the reagent is hottest. These bubbles, when they reach a size thatcan no longer cling to the surface of the microscope slide, will detachand rise to the cooler upper layers of the heated reagent. The bubbleswill then burst at the surface of the reagent to release the gaseousphase to the atmosphere. This energy release cools the upper layer ofthe reagent. This property allows only the lower layer to reach theboiling point, and the remainder of the reagent only reachestemperatures below the boiling point of the reagent due to theevaporative heat loss, and subsequent reduction of the net temperatureof the reagent. The reagent slowly loses its volume to evaporation andnever reaches a constant vigorous boiling condition necessary for mostantigen retrieval protocols.

In contrast, as occurs during the process of the present invention, thereaction compartment is sealed or substantially sealed, such that thereagent would quickly come to equilibrium in a boiling state throughoutthe layers of reagent and would maintain a vigorous constant boilingcondition and would build up pressure inside the closed reactioncompartment. If not regulated, the pressure could exceed safe levels andthe reaction compartment could eventually fail under pressure. To havethe benefits of a constant vigorous boiling effect of the reagent on theslide, the evaporative heat loss must be regulated by enclosing thereaction compartment sufficiently so as to release the gaseous phase ata rate that maintains a vigorous boiling condition.

The reagent strips shown in FIGS. 39-78 comprise a plurality of capsulessized to contain various amounts of reagents, fluids, or buffers, forexample, from 10 μl to 2-5 ml. The capsules can contain reagents such astains, probes, rinses, antibodies, buffers, chemicals or solvents, andthe reagent strip preferably has at least one vent, slot, or window.Each vent may be preferably from 10 μm to 20 mm in diameter and extendsbetween an upper surface and a lower surface of the reagent strip. Thereare typically from one to twenty vents in each reagent strip but theremay be more in other embodiments. A reagent strip may be constructedwith only a single capsule for dispensing an antigen recovery buffer orother reagent.

The number and diameters of vents and slots in the reagent strips can bevaried depending on the types of reagents and antigen recovery buffersused and the amount of pressure, steam, vapor or gases which are likelyto be released during the process of heating the antigen recover bufferapplied to the microscope slide.

When the vent is a slot or a slit or window, rather than a “hole”, thevent may be from 10 μm to 10 mm wide, for example. There may betypically from one to twenty vents in a reagent strip but may be more inother embodiments. The vents or slots are preferably located in aposition of the reagent strip which is between adjacent capsules or tothe sides of capsules. Preferably any one of the reagent dispensingstrips of the present invention contemplated herein comprises only 1 to25 reagent containers, only 1-20 reagent containers, only 1-15 reagentcontainers or only 1-10 reagent containers.

Embodiments of FIGS. 79-85

As shown in FIGS. 79-85 in an alternate version of a reaction module ofthe apparatus of the present invention, reaction module 210 is similarto reaction module 110 in comprising a reaction compartment 212 similarto reaction compartment 112, a slide support element 214 similar toslide support element 114, and a reagent strip support device 216similar to reagent strip support 116. Reaction compartment 212 comprisesa reaction compartment heater 218 for heating the reaction compartment212 and optionally the slide support element 214 when disposed thereinor other gases or liquids therein. The reaction compartment heater 218has leads 220 thereto for connecting to an electric power source (notshown). The reaction compartment 212 further comprises a reagent conduit222 and an injector port orifice 224 for delivering a reagent or othersolution into the reaction compartment 212. The reaction module 210further comprises a reagent strip heater 226 incorporated into thereagent strip support device 216 for heating a reagent strip (such asany of the reagent strips disclosed herein) disposed thereon. Leads 228connect the reagent strip heater 226 to an electric power source (notshown). The reaction module 210 further comprises a reagent conduitheater 230 for heating the reagent conduit 222 thereby functioning toheat a reagent as it passes through the reagent conduit 222 into thereaction compartment 212 merely onto the microscope slide if the reagentis applied when the slide is outside of the reaction compartment 212.Leads 232 connect the reagent conduit heater 230 to an electric powersource (not shown). The slide support element 214 comprises a base 240and, a handle 242, and a front O-ring 244 and a rear O-ring 246 forsealing the base 240 and microscope slide within the reactioncompartment 212. The slide support element 214 further comprises amicroscope slide platform/heater 248 and in operation has a microscopeslide 250 disposed thereon, the microscope slide 250 having an uppersurface 251. The base 240 further comprises a base cavity 252 positionedbelow the slide platform/heater 248 and has a base cavity heater 254positioned therein and connected via lead 256 to an electric powersource (not shown). The base cavity heater 254 functions to heat areagent 258 disposed within the base cavity 252 to a temperaturesufficient to heat the microscope slide 250 and biological specimen andreagent 258 disposed thereon as described elsewhere herein for otherembodiments of the invention. The reagent 258 in one preferredembodiment completely immerses the microscope slide 250 as shown in FIG.79. The reagent strip support device 216 in this embodiment comprises aslot 260 (which may also be included in the reagent strip support device116) therein for enabling a dispenser plunger (i.e., dispenser element)264 to deliver a reagent 262 directly upon the microscope slide 250either when it is positioned within the reaction compartment 212 (FIGS.79, 82, 83) or outside of the reaction compartment (FIGS. 80, 81). Asshown in FIGS. 80, 81, and 84 reagent may be applied to or removed fromthe microscope slide 250 when the microscope 250 slide is positionedoutside of the reaction compartment 212 on the slide support element214. Reagent may be removed from the microscope slide 250 by thedispenser plunger 264 by moving the tip 266 of the dispenser plunger 264over the microscope slide 250 and aspirating the reagent therefrom.Reagent may be delivered to or removed from the microscope slide 250through one or more conduits 268 in the dispenser plunger 264 (FIG. 84).The conduits 268 may function to provide reagents or solutions, toremove reagents (via aspiration for example), or may provide air, gases,or liquids under pressure.

In other embodiments, reaction modules of the present invention may haveany one or any combination of slide heating elements 136 or 248,reaction compartment heater 218, reagent strip heater 226, reagentconduit heater 230, and base cavity heater 254, and when present any ofthe heating systems described herein may function individually andindependently of one another.

As represented in FIG. 85, a plurality of reaction modules 210 arepreferably positioned within a single chamber 282, wherein one or moresuch chambers 282 may be combined to provide a staining apparatus 280having a microprocessor 284 and comprising, e.g., 5 to 50 reactionmodules 210.

In a preferred embodiment, the reaction compartment and/or slide supportelement of a reaction module of the present invention may be exposed tosterilization conditions which may include high heat (e.g., above 100°C., or more preferably above 130° C., and may use steam and/or chemicalsto remove, or denature pathogens or residual chemicals or materials suchas nucleic acids, antibodies, toxins or other proteins which remain inthe reaction compartment and slide support element after the reactionmodule is used. In a preferred embodiment, the reaction compartmentand/or slide support element after heating is quickly cooled to nearroom temperature or to below 50° C. within 3 s, 5 s, 10 s or 20 s forexample to further denature or inactivate residual proteins orsubstances.

In an alternate embodiment of the invention, a plurality of slides areprocessed (either separately or within a common vessel) by applying areagent or solution to the slide and pressurizing the vessel aboveatmospheric pressure to levels as discussed elsewhere herein, whereinthe biological specimens, biochemicals, or other biological entity onthe slide is not subjected to additional heating.

As described elsewhere herein, preferably the slide support element,reaction compartment, reagent strip, reagent strip support device,dispensing element, ports, conduits, mixing jets, pressurizing means,cooling means, aspiration devices, drainage ports, heating devices, andreagent conduits are independently operable and independently movable.

The in situ antigen recovery and staining apparatus of the presentinvention preferably has as one component a device for reading ordetecting an optical character or code which identifies a reagent stripor reagent strip component such as a tile or container.

Various embodiments of the processes of the present invention include,but are not limited to, (1) application of a reagent to a slide usingthe present apparatus, and heating the slide, with or without a step ofpressurizing the reaction compartment, (2) filling the base cavity witha reagent or solution such that it immerses the slide, pre-pressurizingthe reaction compartment, then heating the slide and reagent solution inthe base cavity, (3) filling the base cavity with a reagent or solution,then heating the slide and reagent or solution, withoutpre-pressurization before the heating step, or (4) placing a liquid inthe bottom of the base cavity without the liquid directly touching theslide, then heating the liquid in the base cavity to cause vaporformation which pressurizes the reaction compartment and secondarilyheats the slide and reagent thereon (the slide also may optionally beheated by the slide heater).

Other embodiments of the present invention are shown and described inU.S. Pat. No. 6,534,008; U.S. Ser. No. 10/245,035; U.S. Pat. No.6,855,292; and U.S. Provisionals 60/142,789; 60/684,047; 60/689,386 and60/730,744, the entirety of each of which is hereby expresslyincorporated herein by reference.

While the invention has been described above, and in further detailbelow, in connection with certain preferred embodiments herein so thataspects thereof may be more fully understood and appreciated, it is notintended to limit the invention to these particular embodiments. To thecontrary, it is intended to cover all alternatives, modifications andequivalents as may be included within the scope of the invention asdefined by the appended claims. Thus, these examples and embodiments,which include preferred embodiments, will serve to illustrate thepractice of this invention, it being understood that the particularsshown are by way of example and for purposes of illustrative discussionof preferred embodiments of the present invention only and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of formulation procedures as well as ofthe principles and conceptual aspects of the invention.

EXAMPLES Example 1

-   -   (1) Place microscope slide on slide support element and enclose        within reaction compartment;    -   (2) Add antigen recovery buffer;    -   (3) Set slide heater at 130° C.;    -   (4) Pressure regulator set at 23 psig (259.9 kPa);    -   (5) Antigen recovery buffer reaches 125° C.;    -   (6) Incubate at 125° C. for 10 minutes;    -   (7) Turn off heater and turn on air or liquid cooling system;    -   (8) Cool 5 minutes; and    -   (9) Rinse with buffer and proceed with staining protocol.

Example 2

-   -   (1) Place microscope slide on support element;    -   (2) Enclose microscope slide within individual reaction        compartment;    -   (3) Dispense 1-2 ml of antigen retrieval reagent onto microscope        slide;    -   (4) Close all external ports;    -   (5) Open pressure port to pre-pressurize reaction compartment to        about 25 psig (273.7 kPa);    -   (6) Turn on heat plate to reach about 120° C. on slide;    -   (7) Set pressure regulator to maintain 120° C. temperature by        regulating the reaction compartment's pressure;    -   (8) Reagent reaches a temp of 120° C.;    -   (9) Heating is maintained for 30 minutes at about 120° C.;    -   (10) Turn off heater and turn on air or liquid cooling system;    -   (11) Cool 5-10 minutes;    -   (12) Release pressure to atmospheric pressure;    -   (13) Cool antigen retrieval reagent;    -   (14) Rinse slide with PBS wash buffer; and    -   (15) Proceed with staining protocol.

Example 3

Three mls of antigen recovery buffer present in reaction compartment canbe heated to a particular reaction temperature at a particular pressure,including for example: 100° C. @ 8 psig (156.6 kPa), 106° C. @ 10 psig(170.3 kPa), 110° C. @ 12 psig (184.0 kPa), 115° C. @ 15 psig (204.7kPa), 120° C. @ 16 psig (211.6 kPa), 125° C. @ 23 psig (259.9 kPa), or130° C. @ 30 psig (308.1 kPa), 140° C. @ 40: retrieval buffer after a 60minutes treatment time.

Example 4

Ambient temperature with pressure staining protocol:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent strip or other dispensing        element;    -   4) Pressurize the chamber with a separate gas to desired        pressure (50-100 psig: 446−790.6 kPa);    -   5) Incubate the reagent for a desired time (10-120 minutes);    -   6) Depressurize the chamber by opening the waste port;    -   7) Rinse slide of reagent by rinsing and/or tilting and rinsing        the slide;    -   8) Repeat steps 3-7 until all reagents are dispensed for a        particular protocol and for a desired time.

Example 5

High temperature Antigen Retrieval protocol with pre-pressurization:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent strip or other dispending element        onto the microscope slide;    -   4) Pressurize the chamber with a separate gas to desired        pressure (15-30 psig: 204.7-308.1 kPa);    -   5) Turn on at least one heating element (i.e., slide heater,        chamber heater, cavity heater) and heat to 125° C.;    -   6) Pressure is maintained at 15-20 psig (204.7-239.2 kPa) by the        pressure release valve or heating modulation (i.e., heating        elements turning off and on);    -   7) Incubate reagent at 125° C. for 10-30 minutes;    -   8) Turn heaters off and turn on cooling ducts (liquid or air)        until reagent drops below 50° C.;    -   9) Depressurize the chamber sending condensation and pressure        out the waste port;    -   10) Rinse slide of reagent by rinsing and/or tilting and rinsing        the slide;    -   11) Dispense regent and incubate with or without pressure and/or        with or without heat for a desired time;    -   12) Repeat steps 9-10 until all reagents are dispensed.

Example 6

High temperature Antigen Retrieval protocol without pre-pressurization:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent strip or other dispending element        and fill up the chamber with reagent by totally immersing the        entire slide in reagent (i.e., antigen retrieval reagent);    -   4) Turn on at least one heating element (i.e., slide heater,        chamber heater, cavity heater) and heat to 125° C.;    -   5) Pressure is produced by the reagent boiling;    -   6) Pressure is maintained at 25 psig (273.7 kPa) by the pressure        release valve or heating modulation (i.e., heating elements        turning off and on);    -   7) Reagent is incubated at a temperature of 125° C. for 10-30        minutes;    -   8) Turn heaters off and turn on cooling ducts (liquid or air)        until reagent drops below 50° C.;    -   9) Depressurize the chamber sending condensation, reagent, and        pressure out the waste port;    -   10) Rinse slide or reagent by rinsing and/or tilting and rinsing        the slide;    -   11) Dispense reagent and incubate with or without pressure        and/or with or without heat for a desired time;    -   12) Repeat steps 10-11 until all reagents are dispensed.

Example 7

High temperature Antigen Retrieval protocol—cavity produces steam tomaintain high heat conditions with pressurization:

-   -   1) Place slide on slide support;    -   2) Close chamber to seal slide support to chamber;    -   3) Dispense reagent by reagent strip or other dispending element        onto the microscope slide;    -   4) Add deionized (D.I.) water, or other liquid reagent to the        cavity below the slide (deionized water not contacting the        microscope slide);    -   5) Turn on slide heating element and cavity heaters and heat to        125° C.;    -   6) Pressure is produced by the deionized water boiling in the        cavity and producing steam to heat the reagent on the microscope        slide;    -   7) Pressure is maintained at 25 psig (273.7 kPa) by the pressure        release valve or heating modulation (i.e., heating elements        turning off and on);    -   8) Reagent is incubated at a temperature of 125° C. for 10-60        minutes;    -   9) Turn heaters off and turn on cooling ducts (liquid or air)        until reagent drops below 50° C.;    -   10) Depressurize the chamber sending condensation, deionized        water and pressure out the water port;    -   11) Rinse slide of reagent by rinsing and/or tilting and rinsing        the slide;    -   12) Dispense reagent and incubate with or without pressure        and/or with or without heat for a desired time;    -   13) Repeat steps 10-11 until all reagents are dispensed.

In summary, the invention in one embodiment contemplates an in situantigen recovery and staining apparatus, comprising a plurality ofindependently operable reaction modules with each reaction modulecomprising: a reaction compartment having an inner space, a slidesupport element able to support a microscope slide in a horizontalposition, the slide support element positionable within or adjacent theinner space of the reaction compartment for sealing the microscope slidetherein wherein the reaction compartment is pressurizable (or optimallydepressurizable) to maintain an internal pressure which exceeds (or isbelow) atmospheric pressure, and a dispensing element for dispensing areagent into the reaction compartment while the reaction compartment ispressurized, and may further comprise a heating element for heating themicroscope slide within the reaction compartment.

The present invention contemplates a reaction module, comprising: areaction compartment having an inner space, a slide support element ableto support a microscope slide in a horizontal position, the slidesupport element positionable within or adjacent the inner space of thereaction compartment for sealing the microscope slide therein whereinthe reaction compartment can then be pressurized (or, optionally,depressurized) to maintain an internal pressure which exceeds (or isbelow) atmospheric pressure, and a dispensing element for dispensing areagent into the reaction compartment while the reaction compartment ispressurized. The reaction module may optionally have a heating elementfor heating the microscope slide, and/or a reagent strip support devicefor supporting a reagent strip having a plurality of reagent containerseach of which contains or is able to contain a reagent therein, whereinthe reagent strip support device supports the reagent strip in aposition external to and adjacent the reaction compartment, and thedispensing element may be adapted to engage the reagent containerthereby causing the reagent to be delivered from the reagent containerinto the inner space of the reaction compartment and onto the microscopeslide.

More particularly, the invention contemplates an in situ antigenrecovery and staining apparatus, comprising a plurality of independentlyoperable reaction modules, wherein each reaction module comprises: areaction compartment having an inner space, a slide support element ableto support a microscope slide in a horizontal position, the slidesupport element positionable within or adjacent the inner space of thereaction compartment for sealing the microscope slide therein whereinthe reaction compartment can then be pressurized (or, optionally,depressurized) to maintain an internal pressure which exceeds (or isbelow) atmospheric pressure, a heating element for heating themicroscope slide, a reagent strip support device for supporting areagent strip having a plurality of reagent containers each of whichcontains or is able to contain a reagent therein, wherein the reagentstrip support device supports the reagent strip in a position externalto and adjacent the reaction compartment, and a dispensing element forengaging the reagent container thereby causing the reagent to bedelivered from the reagent container into the inner space of thereaction compartment and onto the microscope slide, and wherein each ofthe reaction compartments of the plurality of reaction modules isindividually and independently pressurizable (or, optionally,depressurizable) and wherein each of the heating elements of theplurality of reaction modules is individually and independentlyheatable.

In the in situ antigen recovery and staining apparatus, the reactioncompartment may be pressurizable before, during, or after the heatingelement heats the microscope slide, the heating element may be acomponent of the slide support element and may be positionable directlybeneath the microscope slide, the reaction compartment may have acylindrical, tubular shape wherein the slide support element has acylindrical shape, or the reaction compartment may have a rectangularshape, such that the slide support element has a rectangular shape.

In the in situ antigen recovery and staining apparatus, the slidesupport element of each reaction module may be independently movable inrelation to each other slide support element, the reagent strip of eachreaction module may be independently movable in relation to each otherreagent strip, the reaction compartment of each reaction module may beindependently movable in relation to each other reaction compartment,and the dispensing element of each reaction module may be independentlymovable in relation to each other dispensing element, the reactioncompartment is preferably pressurizable to maintain a pressure aboveatmospheric pressure, such as 0 to 350 psig (101.3-2514 kPa), to apressure of 1 to 100 psig (108.2-790.6 kPa), to a pressure of 5 to 50psig (135.8−446.0 kPa), or to a pressure of 10 to 40 psig (170.3-377.0kPa), or is depressurizable to maintain a pressure below atmosphericpressure to a level as low as 100 kPa to 10 kPa to 1 kPa to 100 Pa to 10Pa to 1 Pa to 0.1 Pa.

In the in situ antigen recovery and staining apparatus, the reagentdisposed onto or about the microscope slide may be heated to atemperature of 25° C. to 37° C., 37° C. to 56° C., 56° C. to 85° C., 85°C. to 100° C., 100° C. to 125° C., 125° C. to 135° C., 135° C. to 150°C., 150° C. to 175° C., 175° C. to 200° C., 200° C. to 225° C., 225° C.to 250° C., 250° C. to 275° C., 275° C. to 300° C., 300° C. to 325° C.,or 325° C. to 350° C. When the slide support element of the reactionmodule is movable, the reaction compartment may be stationary ormovable, and the reagent strip support device may be stationary ormovable, when the slide support element of the reaction module isstationary, the reaction compartment is movable, and the reagent stripsupport device may be stationary or movable. When the slide supportelement of the reaction module is movable or stationary, and thereaction compartment is movable, and the reagent strip support device ismovable, the reaction compartment may be movable independently of thereagent strip support device. Further, the reagent strip support devicemay be movable in either a forward or reverse direction to carry thereagent strip when loaded thereon in either a forward or reversedirection, and when the reagent strip support device is stationary, thereagent strip may be movable in either a forward or reverse directionwhen loaded thereon. The reagent strip support device and the reactioncompartment are connected to each other, or not connected. The reactionmodule may comprise at least one of an air duct for pressurizing thereaction compartment or causing mixing of the reagent on the slide, or acooling duct for enhancing the rate of cooling of the heating elementafter heating, a supply port for delivering a liquid to the slidesupport element, and a drainage duct for removing reagents supplied tothe microscope slide. The apparatus may comprise a reagent conduit inthe reaction module for enabling delivery of reagent from the reagentstrip into the reaction compartment, a heating device disposed about thereagent conduit for heating the reagent delivered therethrough, aheating device for heating the reaction compartment, and a heatingdevice in the reagent strip support device for heating the reagent stripor portions thereof.

The slide support element of the apparatus may have a cavity in aposition below the microscope slide for containing a quantity ofsolution and the cavity may have a cavity heater for heating thesolution within the cavity. The dispensing element may be operableindependently of the reagent strip support device, and the dispensingelement preferably functions to cause expulsion of reagent from areagent container of the reagent strip and to dispense a reagent orsolution from a reagent or solution source remote from the reagentstrip. The slide support element may receive reagent from the reagentstrip or reagent or solution from a remote source when the slide supportelement is disposed inside or outside of the reaction compartment. Thedispensing element is preferably able to apply suction, or is able toapply liquid, air, or gas under pressure. The slide support element maybe encloseable within the reaction compartment by moving the slidesupport element into the reaction compartment or by moving the reactioncompartment about the slide support element. The slide support elementmay be tiltable to allow drainage of reagent or solution from themicroscope slide. The plurality of reaction modules can be assembledinto at least one chamber to form a reaction apparatus. Each slidesupport element, reagent strip support device, dispensing element, andreaction compartment of the apparatus is preferably separatelyreplaceable or exchangeable, and the reaction module preferably hasmeans for releasing pressure from or regulating pressure within thereaction compartment.

The present invention also contemplates a reconfigurable reagentdispensing strip, comprising a plurality of reagent modules, eachreagent module comprising a tile and a reagent container securedthereto, each reagent module adapted to be attachable to and detachablefrom an adjacent reagent module such that once the plurality of reagentmodules are attached together in a first sequence, one or more of thereagent modules can be detached and reattached to reconfigure theplurality of reagent modules in a second sequence different from thefirst sequence. The reconfigurable reagent dispensing strip may have aconnecting link for connecting adjacent reagent modules, and an injectorfor enabling a reagent within the reagent container to be dispensed fromthe reagent container, and the reagent container may be removable fromthe tile. Further, at least one of the reagent containers contains areagent selected from the group consisting of antigen retrievalreagents, RNA and DNA probes, citrate buffer, EDTA, TRIS, PBS, with orwithout surfactants or detergents like SDS, Tween, Brij, ionic and nonionic detergents, and silicone additives, rinse buffers,immunohistochemical reagents, histochemical reagents, in-situhybridization reagents, PCR reagents, coverslipping reagents, siliconeoils, mineral oils, detection reagents and processing reagents, liquidreagents, reconstituted dry reagents, biological reagents and aqueousand non-aqueous reagents, and deparaffinizing compositions of water withone or more silicone surfactants or silicone additives.

Alternatively, the reconfigurable reagent dispensing strip may comprisea base, having a plurality of container platforms, and a plurality ofreagent containers, with each container platform having a reagentcontainer secured thereto, wherein each reagent container is adapted tobe attachable to and detachable from the container platform such thatonce the plurality of reagent containers are attached together in afirst sequence, one or more of the reagent containers can be detachedand reattached to a different container platform to reconfigure theplurality of reagent containers in a second sequence different from thefirst sequence, thereby forming a reconfigured reagent dispensing strip.The reagent container may be positioned upon a tile which is detachablefrom the base. The reagent container or container platform may furthercomprise an injector for enabling a reagent within the reagent containerto be dispensed from the reagent container. At least one of the reagentcontainers contains a reagent selected from the group consisting ofantigen retrieval reagents, RNA and DNA probes, citrate buffer, EDTA,TRIS, PBS, with or without surfactants or detergents like SDS, Tween,Brij, ionic and non ionic detergents, and silicone additives, rinsebuffers, immunohistochemical reagents, histochemical reagents, in-situhybridization reagents, PCR reagents, coverslipping reagents, siliconeoils, mineral oils, detection reagents and processing reagents, liquidreagents, reconstituted dry reagents, biological reagents and aqueousand non-aqueous reagents, and deparaffinizing compositions of water withone or more silicone surfactants or silicone additives.

Alternatively, the reconfigurable reagent dispensing strip may comprisea plurality of reagent modules, each reagent module comprising a tileand a reagent container secured thereto, wherein the tiles are initiallyconstructed in a unitary, integral configuration and each tile isadapted to be attachable to and detachable from an adjacent tile suchthat the reagent modules are connected in a first sequence, and whereinwhen one or more of the tiles is detached, the one or more tiles can bereattached to reconfigure the plurality of reagent modules in a secondsequence different from the first sequence, and may further comprise aconnecting link for re-connecting tiles of adjacent reagent modules. Thereagent module may further comprise an injector for enabling a reagentwithin the reagent container to be dispensed from the reagent container,and the reagent container may be removable from the tile. Further, atleast one of the reagent containers contains a reagent selected from thegroup consisting of antigen retrieval reagents, RNA and DNA probes,citrate buffer, EDTA, TRIS, PBS, with or without surfactants ordetergents like SDS, Tween, Brij, ionic and non ionic detergents, andsilicone additives, rinse buffers, immunohistochemical reagents,histochemical reagents, in-situ hybridization reagents, PCR reagents,coverslipping reagents, silicone oils, mineral oils, detection reagentsand processing reagents, liquid reagents, reconstituted dry reagents,biological reagents and aqueous and non-aqueous reagents, anddeparaffinizing compositions of water with one or more siliconesurfactants or silicone additives.

In another embodiment, the present invention contemplates a method oftreating a microscope slide, comprising: providing a plurality ofindependently operable reaction modules, each reaction modulecomprising: a reaction compartment having an inner space, a slidesupport element able to support a microscope slide in a horizontalposition, the slide support element positionable within or adjacent theinner space of the reaction compartment for sealing the microscope slidetherein, and a dispensing element for dispensing a reagent into thereaction compartment, then disposing the microscope slide onto the slidesupport element, positioning the microscope slide within the reactioncompartment, pressurizing the reaction compartment to maintain aninternal pressure which exceeds atmospheric pressure, and actuating thedispensing element to cause the reagent to be delivered into thereaction compartment while the reaction compartment is pressurized andwherein the reagent is delivered at a pressure which exceeds thepressure within the reaction compartment, and optionally heating themicroscope slide and reagent within the reaction compartment. The methodmay comprise providing a plurality of independently operable reactionmodules, each reaction module comprising a reaction compartment havingan inner space, a slide support element able to support a microscopeslide in a horizontal position, the slide support element positionablewithin or adjacent the inner space of the reaction compartment forsealing the microscope slide therein, and a dispensing element fordispensing a reagent into the reaction compartment, then disposing themicroscope slide onto the slide support element, positioning themicroscope slide within the reaction compartment and enclosing themicroscope slide therein, actuating the dispensing element to cause thereagent to be delivered into the reaction compartment, and pressurizingthe reaction compartment to maintain an internal pressure which exceedsatmospheric pressure, or, optionally, depressurizing the reactioncompartment below atmospheric pressure, and, optionally, heating themicroscope slide and reagent within the reaction compartment.

The method of the invention may comprise providing a reaction modulecomprising: a reaction compartment having an inner space, and a slidesupport element able to support a microscope slide in a horizontalposition, the slide support element positionable within or adjacent theinner space of the reaction compartment for sealing the microscope slidetherein, then disposing the microscope slide onto the slide supportelement, positioning the slide support element and microscope slidethereon within the reaction compartment, pressurizing the reactioncompartment to maintain an internal pressure which exceeds atmosphericpressure, and disposing a reagent onto the microscope slide while thereaction compartment is pressurized, and optionally heating themicroscope slide and the reagent thereon.

The method may comprise providing a plurality of independently operablereaction modules, each reaction module comprising: a reactioncompartment having an inner space, a slide support element able tosupport a microscope slide in a horizontal position, the slide supportelement positionable within or adjacent the inner space of the reactioncompartment for sealing the microscope slide therein, and a dispensingelement for dispensing a reagent into the reaction compartment, thendisposing the microscope slide onto the slide support element,positioning the microscope slide within the reaction compartment,depressurizing the reaction compartment to maintain an internal pressurewhich is less than atmospheric pressure, and actuating the dispensingelement to cause the reagent to be delivered into the reactioncompartment while the reaction compartment is depressurized.

The method may comprise providing a plurality of independently operablereaction modules, each reaction module comprising: a reactioncompartment having an inner space, a slide support element able tosupport a microscope slide in a horizontal position, the slide supportelement positionable within or adjacent the inner space of the reactioncompartment for sealing the microscope slide therein, and a dispensingelement for dispensing a reagent into the reaction compartment, thendisposing the microscope slide onto the slide support element,positioning the microscope slide within the reaction compartment andenclosing the microscope slide therein, actuating the dispensing elementto cause the reagent to be delivered into the reaction compartment, anddepressurizing the reaction compartment to maintain an internal pressurewhich is less than atmospheric pressure.

Preferably the invention comprises a method of treating a microscopeslide, comprising, providing a plurality of independently operablereaction modules, each reaction module comprising: a reactioncompartment having an inner space, a slide support element able tosupport a microscope slide in a horizontal position, the slide supportelement positionable within or adjacent the inner space of the reactioncompartment for sealing the microscope slide therein, a heating elementfor heating the microscope slide, a reagent strip support device forsupporting a reagent strip having a plurality of reagent containers eachof which contains or is able to contain a reagent therein, wherein thereagent strip support device supports the reagent strip in a positionexternal to and adjacent the reaction compartment, and a dispensingelement for engaging the reagent container thereby causing the reagentto be delivered from the reagent container into the inner space of thereaction compartment and onto the microscope slide, and wherein each ofthe reaction compartments of the plurality of reaction modules isindividually and independently pressurizable (or, optionally,depressurizable) and wherein each of the heating elements of the slidesupport elements of the plurality of reaction modules is individuallyand independently heatable, then disposing the microscope slide onto theslide support element, positioning the slide support element andmicroscope slide thereon within the reaction compartment, placing thereagent strip onto the reagent strip support device, actuating thedispensing element to cause the reagent to be delivered onto themicroscope slide, actuating the heating element to heat the slide,pressurizing the reaction compartment to maintain an internal pressurewhich exceeds atmospheric pressure, and removing the reagent from themicroscope slide.

In the method, the step of pressurizing (or depressurizing) the reactioncompartment may occur before, during, or after the heating of themicroscope slide by the heating element. The reaction compartment mayhave a cylindrical, tubular shape for enhancing pressure distributionwithin the reaction compartment. The slide support element of eachreaction module may be moved independently in relation to each otherslide support element, the reagent strip of each reaction module may bemoved independently in relation to each other reagent strip, and thedispensing element of each reaction module may be moved independently inrelation to each other dispensing element. The reaction compartment maybe pressurized to a pressure of above 0 to 350 psig (101.3-2514 kPa), toa pressure of 1 to 100 psig (108.2-790.6 kPa), to a pressure of 5 to 50psig (135.8-446.0 kPa), or to a pressure of 10 to 40 psig (170.3-377.0kPa). The reaction compartment may be depressurized to maintain apressure below atmospheric pressure to a level as low as 100 kPa to 10kPa to 1 kPa to 100 Pa to 10 Pa to 1 Pa to 0.1 Pa. The reagent disposedonto or about the microscope slide may be heated to a temperature of 25°C. to 37° C., 37° C. to 56° C., 56° C. to 85° C., 85° C. to 100° C.,100° C. to 125° C., 125° C. to 135° C., 135° C. to 150° C., 150° C. to175° C., 175° C. to 200° C., 200° C. to 225° C., 225° C. to 250° C.,250° C. to 275° C., 275° C. to 300° C., 300° C. to 325° C., to 325° C.to 350° C. The step of positioning the slide support element maycomprise moving the slide support element of the reaction module intothe reaction compartment while the reaction compartment is stationary,or the step of positioning the slide support element may comprise movingthe slide support element of the reaction module and moving the reactioncompartment. The reagent strip may be positioned in a dispensingposition by moving the reagent strip support device thereby moving thereagent strip to the dispensing position, or by moving the reagent stripwhile the reagent strip support device is stationary. The method maycomprise moving the slide support element of the reaction module, movingthe reaction compartment is movable, and moving the reagent stripsupport device, wherein the reaction compartment is movableindependently of the reagent strip support device.

The step of positioning the slide support element may comprisemaintaining the slide support element of the reaction module stationary,and moving the reaction compartment to enclose the slide supportelement. The reaction compartment may be movable independently of thereagent strip support device and wherein the reagent strip supportdevice may be movable independently of the reaction compartment. Thereagent strip support device may be moved in either a forward or reversedirection to carry the reagent strip in either a forward or reversedirection. The reagent strip support device may be maintained stationaryand the reagent strip thereon may be moved in either a forward orreverse direction. The reaction module may comprise at least one of anair duct for pressurizing the reaction compartment or causing mixing ofthe reagent on the slide, a cooling duct for enhancing the rate ofcooling of the heating element after heating, a supply port fordelivering a liquid to the slide support element, and a drainage ductfor removing reagents supplied to the microscope slide.

The method may comprise delivering reagent from the reagent strip intothe reaction compartment via a reagent conduit in the reaction module,heating the reagent conduit for heating the reagent deliveredtherethrough, heating the reaction compartment, heating the reagentstrip or portions thereof using a heating device in the reagent stripsupport device, dispensing a solution in a cavity in the slide supportelement below the microscope slide and heating the solution in thecavity. The dispensing element may be operable independently of thereagent strip support device. The method may comprise using thedispensing element to dispense a reagent or solution from a reagent orsolution source remote from the reagent strip, applying reagent from thereagent strip or reagent or solution from a remote source when the slidesupport element is disposed inside or outside of the reactioncompartment, and/or applying suction, or liquid, air, or gas underpressure to the microscope slide via the dispensing element to causeremoval of a reagent or solution from the microscope slide. The step ofpositioning the slide support element within the reaction compartmentmay occur by moving the slide support element into the reactioncompartment or by moving the reaction compartment about the slidesupport element thereby enclosing the slide support element within thereaction compartment, and may comprise the step of tilting the slidesupport element to allow drainage of reagent or solution from themicroscope slide.

While the invention has been described herein in connection with certainembodiments so that aspects thereof may be more fully understood andappreciated, it is not intended that the invention be limited to theseparticular embodiments. On the contrary, it is intended that allalternatives, modifications and equivalents are included within thescope of the invention as defined by the appended claims. Thus theexamples and embodiments described herein, which include preferredembodiments, will serve to illustrate the practice of this invention, itbeing understood that the particulars shown are by way of example andfor purposes of illustrative discussion of preferred embodiments of thepresent invention only and are presented in the cause of providing whatis believed to be the most useful and readily understood description ofprocedures as well as of the principles and conceptual aspects of theinvention.

Changes may be made in the construction and the operation of the variouscomponents, elements and assemblies described herein or in the steps orthe sequence of steps of the methods described herein without departingfrom the spirit and scope of the invention as defined in the followingclaims.

1. An automated apparatus for treating biological specimens, a pluralityof reaction compartments, each having an inner space; a plurality ofindependently movable slide support elements, each able to support amicroscope slide, and wherein each slide support element is movable to aposition within the reaction compartment corresponding thereto; sealingmeans corresponding to each reaction compartment for forming apressure-tight seal for sealing the inner space of the reactioncompartment; pressurizing means for pressurizing the inner space of eachreaction compartment after the inner space has been sealed by thesealing means wherein each reaction compartment is pressurizableindependently of each other reaction compartment; and a microprocessorable to control the pressurizing means to individually pressurize thesealed inner space of the reaction compartment to a pressure level aboveatmospheric pressure.
 2. The apparatus of claim 1 wherein at least aportion of the reaction compartment has a tubular shape, and at least aportion of the slide support element has a tubular shape.
 3. Theapparatus of claim 1 wherein at least a portion of the reactioncompartment has a rectangular shape, and at least a portion of the slidesupport element has a rectangular shape.
 4. The apparatus of claim 1wherein each reaction compartment is movable independently of each otherreaction compartment.
 5. The apparatus of claim 1 wherein each reactioncompartment is operable independently of each other reactioncompartment.
 6. The apparatus of claim 1 wherein the reactioncompartment is sized to contain a single microscope slide.
 7. Theapparatus of claim 1 wherein one of the reaction compartment and theslide support element corresponding thereto is independently movablewhile the other is stationary.
 8. The apparatus of claim 1 wherein boththe reaction compartment and the slide support element correspondingthereto are movable independently of each other.
 9. The apparatus ofclaim 1 comprising a chamber which contains the plurality of reactioncompartments and wherein each independently movable slide supportelement is independently movable to a position for placement of amicroscope slide thereon or for removal of a microscope slide therefrom.10. The apparatus of claim 1 wherein the pressure level aboveatmospheric pressure in the sealed inner space can be maintained withoutheating a microscope slide, or a biological specimen, and/or a reagentin the sealed inner space.
 11. The apparatus of claim 1 wherein thepressure level in the sealed inner space of the reaction compartment canbe maintained at a level in a range of 1 to 350 psig.
 12. The apparatusof claim 1, wherein the reaction compartment can be pressurized to thepressure level above atmospheric pressure before, during, or after amicroscope slide is heated within the reaction compartment.
 13. Theapparatus of claim 1, wherein the reaction compartment can bepressurized to the pressure level above atmospheric pressure before,during, or after a reagent is dispensed into the reaction compartment bya reagent dispensing system.
 14. The apparatus of claim 1 wherein thereaction compartment is sized to contain a volume of solution in a rangeof 0.1 ml to 100 ml.
 15. The apparatus of claim 1 wherein the sealedinner space of each reaction compartment can be separately sterilized.16. The apparatus of claim 1 wherein the slide support element has acavity for containing a solution.
 17. The apparatus of claim 1 whereinthe slide support element can be rotated, turned, or moved inside thecorresponding reaction compartment.
 18. The apparatus of claim 1 whereinthe sealing means is constructed of plastic, polymer, thermoplastic,resin, ceramic, rubber, metal, glass, or a composite.
 19. The apparatusof claim 1 wherein the sealing means is a ring, a seal, an inflatableseal, or comprises mating surfaces of the slide support element and thereaction compartment.
 20. The apparatus of claim 1 comprising a heatingdevice which can heat a microscope slide on the slide support elementand/or a reagent disposed onto or about a microscope slide on the slidesupport element.
 21. The apparatus of claim 20 wherein the heatingdevice is able to heat a microscope slide or a reagent to a temperatureof at least one of 25° C. to 37° C., 37° C. to 56° C., 56° C. to 85° C.,85° C. to 100° C., 100° C. to 125° C., 125° C. to 135° C., 135° C. to150° C., 150° C. to 175° C., 175° C. to 200° C., 200° C. to 225° C.,225° C. to 250° C., 250° C. to 275° C., 275° C. to 300° C., 300° C. to325° C., and 325° C. to 350° C. when the sealed inner space of thereaction compartment is pressurized.
 22. The apparatus of claim 1wherein the pressurizing means comprises a heating element in or on theslide support element and/or a heating element disposed within a cavityof the slide support element for heating a solution within the cavity.23. The apparatus of claim 1 wherein the pressurizing means comprises aheating device positioned within a wall of the reaction compartment. 24.The apparatus of claim 1 wherein the pressurizing means comprises a ductthrough which a pressurized gas is introduced into the inner space ofthe reaction compartment from an external source.
 25. The apparatus ofclaim 1 wherein each pressurizing means is movable and/or operableindependently of each other pressurizing means for each reactioncompartment.
 26. The apparatus of claim 1 wherein the microprocessor isprogrammable.
 27. The apparatus of claim 1 wherein the microprocessor isprogrammable to automatically control movement of the movable slidesupport element and/or movement of the reaction compartment.
 28. Theapparatus of claim 1 comprising a plurality of independently operablereaction modules wherein each reaction module comprises one of thereaction compartments, the independently movable slide support elementand sealing means corresponding to said reaction compartment, andheating means.
 29. The apparatus of claim 28 wherein each reactionmodule comprises at least one of a gas duct for pressurizing thereaction compartment or for causing mixing of a reagent, a cooling ductfor enhancing the rate of cooling of the heating means after heating, asupply port for delivering a liquid, and a drainage duct for removingreagents.
 30. The apparatus of claim 1 comprising a reagent dispensingsystem for dispensing a reagent or solution to a microscope slide oneach slide support element.
 31. The apparatus of claim 30 wherein thereagent dispensing system comprises a plurality of dedicatedindependently movable and/or independently operable reagent dispensingelements each associated with a corresponding reaction compartment. 32.The apparatus of claim 30 wherein the reagent dispensing system providesthe reagent or solution via a reagent device having one or more reagentcontainers, and/or via a remote source, wherein the slide supportelement may be disposed inside or outside of the reaction compartmentwhen the reagent or solution is provided.
 33. The apparatus of claim 30wherein the reagent dispensing system comprises a plurality of reagentstrip support devices positioned adjacent the reaction compartments forsupporting each reagent strip and wherein the reagent strip supportdevice functions to receive, support, and eject the reagent strip. 34.The apparatus of claim 30 wherein the reagent dispensing system is ableto independently apply suction, aspiration, vacuum, liquid, air, or gasto the microscope slide when the microscope slide is disposed within oroutside of the reaction compartment.
 35. The apparatus of claim 30wherein the microprocessor is programmable to automatically control thereagent dispensing system to dispense a reagent onto the microscopeslide in the reaction compartment while the pressure level in the sealedinner space of the reaction compartment is at the pressure level aboveatmospheric pressure.
 36. The apparatus of claim 30 wherein the reagentdispensing system comprises a plurality of movable reagent dispensingelements and wherein the microprocessor is programmable to automaticallycontrol movement of each movable reagent dispensing element andautomatically control the dispensing of a reagent therefrom while thepressure level in the sealed inner space of the reaction compartment isat the pressure level above atmospheric pressure.
 37. The apparatus ofclaim 30 wherein the reagent dispensing system is able to dispense thereagent or solution into the reaction compartment before, during orafter the sealed inner space of the reaction compartment is pressurized.38. The apparatus of claim 30 wherein the reagent dispensing system isable to dispense a reagent or solution into the sealed inner space whilethe sealed inner space of the reaction compartment is pressurized. 39.The apparatus of claim 1 wherein each reaction compartment comprises aport through which a reagent or a solution can be dispensed into thereaction compartment while the pressure level within the sealed innerspace is being maintained above atmospheric pressure.
 40. The apparatusof claim 1 wherein the slide support element is manually movable into orout of the reaction compartment.
 41. The apparatus of claim 1 comprisinga power source comprising AC, DC, solar, pneumatic, or steam.
 42. Theapparatus of claim 1 wherein each of the plurality of reactioncompartments comprises a plurality of processing components which areindependently movable.
 43. The apparatus of claim 1 further comprising aplurality of independently operable aspiration devices.
 44. Theapparatus of claim 1 further comprising a plurality of independentlymovable aspiration devices.
 45. The apparatus of claim 1 wherein each ofthe plurality of reaction compartments comprises a plurality ofprocessing components which are automatically movable.
 46. The apparatusof claim 1 wherein the microprocessor is programmed to control thepressurizing means to individually pressurize the sealed inner space ofthe reaction compartment to a pressure level in a range of 5 to 50 psig.47. The apparatus of claim 1 wherein the slide support element ismovable to a position outside of the inner space of the reactioncompartment for placement and/or removal of a microscope slide on theslide support element.
 48. The apparatus of claim 1 wherein the slidesupport element is movable to a position outside of the inner space ofthe reaction compartment for dispensing a reagent or solution onto amicroscope slide on the slide support element.
 49. The apparatus ofclaim 1 wherein the slide support element is movable to a positionoutside of the inner space of the reaction compartment for removal of areagent or solution from a microscope slide on the slide supportelement.
 50. The apparatus of claim 1 wherein the slide support elementis movable to a treatment position in which position a biologicalspecimen on a microscope slide on the slide support element is withinthe inner space of the reaction compartment while another slide supportelement is in a position outside of an inner space of another reactioncompartment.
 51. The apparatus of claim 1 wherein the slide supportelement is movable from a removal or placement position outside of thereaction compartment associated therewith into a treatment position inwhich a biological specimen on a microscope slide thereon is positionedwithin the inner space of the reaction compartment while another slidesupport element is in a treatment position in which a biologicalspecimen on a microscope slide thereon is positioned within an innerspace of a reaction compartment associated therewith.
 52. The apparatusof claim 1 wherein the pressurizing means comprises a heating elementfor pressurizing the inner space of the reaction compartment bymodulation of the heating element.
 53. The apparatus of claim 1 whereinthe microprocessor is able to control the pressurizing means toindividually pressurize the sealed inner space of the reactioncompartment to a pressure level of at least one of 1 psig, 5 psig, 8psig, 10 psig, 12 psig, 15 psig, 16 psig, 19 psig, 20 psig, 23 psig, 25psig, 30 psig, 40 psig, 50 psig, 100 psig, and 350 psig.
 54. Theapparatus of claim 1 wherein the microprocessor is able to control thepressurizing means to individually pressurize and maintain the sealedinner space of the reaction compartment at a pressure level of 15 psig.55. The apparatus of claim 1 wherein the microprocessor is able tocontrol the pressurizing means to maintain the sealed inner space of thereaction compartment at a pressure level in a range of 1 to 350 psig, 1to 100 psig, 5 to 50 psig, or 10-40 psig.
 56. The apparatus of claim 1wherein the microprocessor is able to control the pressurizing means tomaintain the sealed inner space of the reaction compartment at apressure level of at least one of 1 psig, 5 psig, 8 psig, 10 psig, 12psig, 15 psig, 16 psig, 19 psig, 20 psig, 23 psig, 25 psig, 30 psig, 40psig, 50 psig, 100 psig, and 350 psig.
 57. The apparatus of claim 1comprising a plurality of chambers which are stackable or are joinedlinearly.
 58. The apparatus of claim 1 comprising a plurality ofprocessing components which are independently movable and activatable bythe microprocessor.
 59. The apparatus of claim 1 comprising processingcomponents selected from the group of reagent dispensers, reagent stripsupport devices, reagent strips, aspiration devices, reagent conduits,rinse ports, vacuum ports, drainage ports, mixing jets, air coolingducts, liquid cooling ducts, pressure ports, waste ports, and heatingdevices.
 60. The apparatus of claim 1 comprising heating means able toheat a reagent or solution in the sealed inner space to a temperaturewhich exceeds 100° C.
 61. The apparatus of claim 1 wherein a reagent orsolution can be disposed on a biological specimen in the sealed innerspace before pressurization of the sealed inner space of the reactioncompartment then can be heated to a temperature which exceeds 100° C.,after the sealed inner space has been pressurized.
 62. The apparatus ofclaim 1 wherein the reaction compartment is able to contain at least aportion of a microscope slide disposed on the slide support element. 63.The apparatus of claim 1 wherein a reagent or solution can be disposedon a biological specimen on a microscope slide on the slide supportelement in an amount in a range of 0.1 to 4 ml.
 64. An automatedapparatus for treating biological specimens, a plurality of reactioncompartments, each having an inner space; a plurality of independentlymovable slide support elements, each able to support a microscope slide,and wherein each slide support element is movable to a position withinor adjacent the reaction compartment corresponding thereto and can beturned within the reaction compartment corresponding thereto; sealingmeans corresponding to each reaction compartment for forming apressure-tight seal for sealing the inner space of the reactioncompartment; pressurizing means for pressurizing the inner space of eachreaction compartment after the inner space has been sealed by thesealing means wherein each reaction compartment is pressurizableindependently of each other reaction compartment; and a microprocessorable to control the pressurizing means to individually pressurize thesealed inner space of the reaction compartment to a pressure level aboveatmospheric pressure.
 65. The apparatus of claim 64 wherein eachreaction compartment is movable independently of each other reactioncompartment.
 66. The apparatus of claim 64 wherein each reactioncompartment is operable independently of each other reactioncompartment.
 67. The apparatus of claim 64 wherein the reactioncompartment is sized to contain a single microscope slide.
 68. Theapparatus of claim 64 wherein the heating device is able to heat amicroscope slide or a reagent to a temperature of at least one of 25° C.to 37° C., 37° C. to 56° C., 56° C. to 85° C., 85° C. to 100° C., 100°C. to 125° C., 125° C. to 135° C., 135° C. to 150° C., 150° C. to 175°C., 175° C. to 200° C., 200° C. to 225° C., 225° C. to 250° C., 250° C.to 275° C., 275° C. to 300° C., 300° C. to 325° C., and 325° C. to 350°C. when the sealed inner space of the reaction compartment ispressurized.
 69. The apparatus of claim 64 comprising a chamber whichcontains the plurality of reaction compartments and wherein eachindependently movable slide support element is movable to a position forplacement of a microscope slide thereon or for removal of a microscopeslide therefrom.
 70. The apparatus of claim 64 wherein the slide supportelement is movable to a position outside of the inner space of thereaction compartment for placement and/or removal of a microscope slideon the slide support element.
 71. The apparatus of claim 64 wherein theslide support element is movable to a treatment position in whichposition a biological specimen on a microscope slide on the slidesupport element is within the inner space of the reaction compartmentwhile another slide support element is in a position outside of an innerspace of another reaction compartment.
 72. The apparatus of claim 64wherein the slide support element is movable from a removal or placementposition outside of the reaction compartment associated therewith into atreatment position in which a biological specimen on a microscope slidethereon is positioned within the inner space of the reaction compartmentwhile another slide support element is in a treatment position in whicha biological specimen on a microscope slide thereon is positioned withinan inner space of a reaction compartment associated therewith.
 73. Theapparatus of claim 64 comprising a plurality of processing componentswhich are independently movable and activatable by the microprocessor.74. The apparatus of claim 64 wherein the microprocessor is programmableto automatically control movement of the movable slide support elementand/or movement of the reaction compartment.
 75. The apparatus of claim64 wherein each of the plurality of reaction compartments comprises aplurality of processing components which are automatically movable. 76.The apparatus of claim 64 comprising processing components selected fromthe group of reagent dispensers, reagent strip support devices, reagentstrips, aspiration devices, reagent conduits, rinse ports, vacuum ports,drainage ports, mixing jets, air cooling ducts, liquid cooling ducts,pressure ports, waste ports, and heating devices.
 77. An automatedapparatus for treating biological specimens, a plurality of reactioncompartments, each having an inner space, wherein at least a portion ofeach reaction compartment has a tubular shape; a plurality ofindependently movable slide support elements, each able to support amicroscope slide, and wherein each slide support element is movable to aposition within or adjacent the reaction compartment correspondingthereto; sealing means corresponding to each reaction compartment forforming a pressure-tight seal for sealing the inner space of thereaction compartment; pressurizing means for pressurizing the innerspace of each reaction compartment after the inner space has been sealedby the sealing means wherein each reaction compartment is pressurizableindependently of each other reaction compartment; and a microprocessorable to control the pressurizing means to individually pressurize thesealed inner space of the reaction compartment to a pressure level aboveatmospheric pressure.
 78. The apparatus of claim 77 wherein eachreaction compartment is movable independently of each other reactioncompartment.
 79. The apparatus of claim 77 wherein each reactioncompartment is operable independently of each other reactioncompartment.
 80. The apparatus of claim 77 wherein the reactioncompartment is sized to contain a single microscope slide.
 81. Theapparatus of claim 77 wherein the heating device is able to heat amicroscope slide or a reagent to a temperature of at least one of 25° C.to 37° C., 37° C. to 56° C., 56° C. to 85° C., 85° C. to 100° C., 100°C. to 125° C., 125° C. to 135° C., 135° C. to 150° C., 150° C. to 175°C., 175° C. to 200° C., 200° C. to 225° C., 225° C. to 250° C., 250° C.to 275° C., 275° C. to 300° C., 300° C. to 325° C., and 325° C. to 350°C. when the sealed inner space of the reaction compartment ispressurized.
 82. The apparatus of claim 77 comprising a chamber whichcontains the plurality of reaction compartments and wherein eachindependently movable slide support element is movable to a position forplacement of a microscope slide thereon or for removal of a microscopeslide therefrom.
 83. The apparatus of claim 77 wherein the slide supportelement is movable to a position outside of the inner space of thereaction compartment for placement and/or removal of a microscope slideon the slide support element.
 84. The apparatus of claim 77 wherein theslide support element is movable to a treatment position in whichposition a biological specimen on a microscope slide on the slidesupport element is within the inner space of the reaction compartmentwhile another slide support element is in a position outside of an innerspace of another reaction compartment.
 85. The apparatus of claim 77wherein the slide support element is movable from a removal or placementposition outside of the reaction compartment associated therewith into atreatment position in which a biological specimen on a microscope slidethereon is positioned within the inner space of the reaction compartmentwhile another slide support element is in a treatment position in whicha biological specimen on a microscope slide thereon is positioned withinan inner space of a reaction compartment associated therewith.
 86. Theapparatus of claim 77 comprising a plurality of processing componentswhich are independently movable and activatable by the microprocessor.87. The apparatus of claim 77 wherein the microprocessor is programmableto automatically control movement of the movable slide support elementand/or movement of the reaction compartment.
 88. The apparatus of claim77 wherein each of the plurality of reaction compartments comprises aplurality of processing components which are automatically movable. 89.The apparatus of claim 77 comprising processing components selected fromthe group of reagent dispensers, reagent strip support devices, reagentstrips, aspiration devices, reagent conduits, rinse ports, vacuum ports,drainage ports, mixing jets, air cooling ducts, liquid cooling ducts,pressure ports, waste ports, and heating devices.
 90. The apparatus ofclaim 77 wherein the slide support element can be rotated, turned, ormoved inside the corresponding reaction compartment.